FGF-5 supported and binding peptides

ABSTRACT

The present invention provides peptides and supported peptides that bind to FGF-5.

The present application claims priority under 35 U.S.C. §119, toco-pending U.S. Provisional Patent Application Ser. No. 60/518,154,filed Nov. 6, 2003, co-pending U.S. Provisional Patent Application Ser.No. 60/520,403, filed Nov. 13, 2003, co-pending U.S. Provisional PatentApplication Ser. No. 60/530,954, filed Dec. 19, 2003, co-pending U.S.Provisional Patent Application Ser. No. 60/531,207, filed Dec. 19, 2003,and co-pending U.S. Provisional Patent Application Ser. No. 60/531,189,filed Dec. 19, 2003.

FIELD OF THE INVENTION

The present invention provides peptides and supported peptides that bindFGF-5 molecules.

BACKGROUND OF THE INVENTION

The Fibroblast Growth Factor (FGF) family is a superfamily of growthfactors containing at least 23 members, many of which are potentregulators of cell proliferation, differentiation and cell function. Allof the FGFs have a conserved 120 amino acid core. Members of the familyshare conserved cysteine residues and 30-50% sequence homology at theamino acid level. The molecular weight of the FGFs ranges from 7 kDa forFGF-1 to 38 kDa for FGF-5. The length of the proteins is from 60 aminoacids in the case of an FGF-1 splice variant to 288 amino acids forFGF-2. Binding to heparin is an essential step required for an FGFfactor to interact with cell surface receptors.

FGF5 is a secreted signaling protein consisting of 268 amino acids witha 17 amino acid signal sequence and a 251 amino acid mature peptide. Thehuman gene also gives rise to a glycosylated alternate splice form thatis 18 kDa in size and 123 amino acid in length. The murine homologue ofFGF-5 was cloned and found to be 84% homologous to the human protein atthe amino acid sequence level. Human FGF-5 consists of three exons andmaps to chromosome 4q21 and cross-reacts with murine FGF-5.

Formation of hair follicles involves a complex series of steps: growth(anagen), regression (catagen), rest (telogen) and shedding (exogen).FGF-5 has been implicated as one of the major drivers of the transitionfrom anagen to catagen in the hair cycle. Expression of FGF-5 isdetected in hair follicles from wild-type mice and is localized to theouter root sheath during the anagen phase. Mice homozygous for apredicted null allele of FGF-5, fgf5neo, have abnormally long hair (See,Hebert et al., Cell 78: 1017-25 [1994]). The phenotype appears identicalto that of mice homozygous for the spontaneous mutation angora (go).Recently, partial FGF-5 sequences, FGF5S, thought to compete with FGF-5in binding to the receptor have been identified, (See, Ito et al., J.Cell Physiol., 197:272-83 [2003]).

The Bowman-Birk protease inhibitor (BBI) is a designation of a family ofstable, low molecular weight trypsin and chymotrypsin enzyme inhibitorsfound in soybeans and various other seeds, mainly leguminous seeds andvegetable materials. BBI comprises a family of disulfide bonded proteinswith a molecular weight of about 8 kD (See e.g., Chou et al., Proc.Natl. Acad. Sci. USA 71:1748-1752 [1974]; Yavelow et al., Proc. Natl.Acad. Sci. USA 82:5395-5399 [1985]; and Yavelow et al., Cancer Res.(Suppl.) 43:2454s-2459s [1983]). BBI has a pseudo-symmetrical structureof two tricyclic domains each containing an independent native bindingloop, the native loops containing binding sites for both trypsin andchymotrypsin (See, Liener, in Summerfield and Bunting (eds), Advances inLegume Science, Royal Bot. Gardens, Kew, England). These binding siteseach have a canonical loop structure, which is a motif found in avariety of serine proteinase inhibitors (Bode and Huber, Eur. J.Biochem., 204:433-451 [1992]). Commonly, as in one of the soybeaninhibitors, one of the native loops inhibits trypsin and the otherinhibits chymotrypsin (See, Chen et al., J. Biol. Chem., 267:1990-1994[1992]; Werner & Wemmer, Biochem., 31:999-1010 [1992]; Lin et al., Eur.J. Biochem., 212:549-555 [1993]; and Voss et al., Eur. J. Biochem.,242:122-131 [1996]) though in other organisms (e.g., Arabidopsis), bothloops are specific for trypsin.

STI inhibits the proteolytic activity of trypsin by the formation of astable stoichiometric complex (See e.g., Liu, Chemistry and NutritionalValue of Soybean Components, In: Soybeans, Chemistry, Technology andUtilization, pp. 32-35, Aspen Publishers, Inc., Gaithersburg, Md.,[1999]). STI consists of 181 amino acid residues with two disulfidebridges and is roughly spherically shaped (See e.g., Song et al., J.Mol. Biol., 275:347-63 [1998]). The trypsin inhibitory loop lies withinthe first disulfide bridge. The Kunitz-type soybean trypsin inhibitor(STI) has played a key role in the early study of proteinases, havingbeen used as the main substrate in the biochemical and kinetic work thatled to the definition of the standard mechanism of action of proteinaseinhibitors.

Eglin C is a small monomeric protein that belongs to the potatochymotrypsin inhibitor family of serine protease inhibitors. Theproteins that belong to this family are usually small (60-90 amino acidresidues in length) and contain no disulfide bonds. Eglin C, however, ishighly resistant to denaturation by acidification or heat regardless ofthe lack of disulfide bonds to help stabilize its tertiary structure.The protein occurs naturally in the leech Hirudo medicinalis.

Despite much research into hair growth and its modulation, a needremains for efficient and effective means to modulate hair growth asdesired.

SUMMARY OF THE INVENTION

The present invention provides peptides and supported peptides that bindFGF-5. In particularly preferred embodiments, the present inventionprovides cosmetic and/or pharmaceutical compounds for modulating hairgrowth. The present invention further provides peptides that blockbinding of FGF wherein the peptide is expressed in a protease-resistantscaffold. In some preferred embodiments, the scaffold is a proteaseinhibitor, such as BBI, STI or Eglin chymotrypsin inhibitor.

In some embodiments, the present invention provides cosmetic and/orpharmaceutical compounds comprising at least one polypeptide or apeptide suitable for modulating hair growth. In some preferredembodiments, the compounds comprise at least one polypeptide.

In alternative preferred embodiments, the compounds comprise at leastone peptide. In some preferred embodiments, the peptide has an aminoacid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6,8, 10, 12, and 14 (See, Table 1). In some preferred embodiments, thecompound has a sequence, the sequence being at least 70%, preferably80%, more preferably 90%, and most preferably 95% homologous to thesequences set forth herein. In some preferred embodiments, thepolypeptide has a molecular weight that is preferably between 500Daltons and 30,000 Daltons, more preferably between 1000 Daltons and10,000 Daltons, and most preferably from 1500 Daltons to 8,000 Daltons.

In some preferred embodiments, modulation comprises treatment of atleast one disease or condition that involves loss of hair. In somepreferred embodiments, the disease or condition is at least one selectedfrom the group consisting of inflammatory alopecias, pseudopelade,scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheicdermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia,alopecia areata, cancer, conditions that affect defective hair fiberproduction, and environmental factors that affect hair production. Insome preferred embodiments, the disease is androgenic alopecia oralopecia areata.

In some preferred embodiments, modulation comprises hair growthinhibition and/or hair removal for treatment of at least one disease orcondition for which decreased hair growth is desirable. In somepreferred embodiments, inhibition and/or removal comprises depilation.

In some preferred embodiments, the invention provides cosmetic and/orpharmaceutical compounds for modulating hair growth comprising at leastone peptide or polypeptide and at least one scaffold, the peptide orpolypeptide being contained in the scaffold, preferably the peptide orpolypeptide being a loop, and most preferably, the loop being closed bya disulfide bond. In preferred embodiments, the peptide or polypeptidebinds to FGF-5. In particularly preferred embodiments, the bindingresults in the blocking of the FGF-5 downstream activity. In somepreferred embodiments, the scaffold is STI, Eglin or BBI. Inparticularly preferred embodiments, the scaffold is BBI. In furtherpreferred embodiments, the peptide or polypeptide comprises apolypeptide.

In additional preferred embodiments, the compound comprises at least onepeptide. In some preferred embodiments, the peptide has an amino acidsequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8,10, 12, and 14 (See, Table). In other preferred embodiments, thecompounds has an amino acid sequence comprising any sequence selectedfrom the group consisting of SEQ ID NOS: 15-43 (See, FIG. 10). In somepreferred embodiments, the compound has a sequence, the sequence beingat least 70%, more preferably 80%, even more preferably 90%, and mostpreferably 95% homologous to the sequences set forth herein. In apreferred embodiment, the polypeptide has a molecular weight that ispreferably between 500 Daltons and 100,000 Daltons, more preferablybetween 500 Daltons and 30,000 Daltons, even more preferably between 500Daltons and 45,000 Daltons, still more preferably between 1000 Daltonsand 12,000 Daltons, and most preferably from 1500 Daltons to 10,000Daltons.

In some preferred embodiments, modulation comprises treatment of atleast one disease or condition that involves loss of hair. In somepreferred embodiments, the disease or condition is at least one selectedfrom the group consisting of inflammatory alopecias, pseudopelade,scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheicdermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia,alopecia areata, cancer, conditions that affect defective hair fiberproduction, and environmental factors that affect hair production. Insome particularly preferred embodiments, the disease is androgenicalopecia or alopecia areata.

In some preferred embodiments, modulation comprises hair growthinhibition and/or hair removal for treatment of at least one disease orcondition for which decreased hair growth is desirable. In somepreferred embodiments, inhibition and/or removal comprises depilation.

In yet further embodiments, the present invention provides cosmeticand/or pharmaceutical compositions comprising at least one polypeptideor peptide, as set forth herein, and a physiologically acceptablecarrier or excipient. Preferably, the compound is present in an amountof about 0.0001% to about 5% by weight based on the total weight of thecomposition. Also preferably, the compound is present in an amount ofabout 0.001% to about 0.5% by weight based on the total weight of thecomposition. The composition may be in the form of an emulsifiedvehicle, such as a nutrient cream or lotion, a stabilized gel ordispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In yet further embodiments, the present invention provides means fordecreasing FGF-5 activity. In some embodiments, the method comprisingapplying an effective amount of at least one of the compounds describedherein to an organism in need thereof.

The present invention provides compositions comprising at least onepeptide selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10,12, and 14, wherein said peptide binds to a fibroblast growth factor. Insome embodiments, the peptide is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, and13. In some preferred embodiments, the peptide is expressed in aprotease resistant scaffold. In some particularly preferred embodiments,the scaffold is a protease inhibitor. In some preferred embodiments, theprotease inhibitor is selected from the group consisting of Bowman-BirkInhibitor, soybean trypsin inhibitor, and Eglin chymotrypsin inhibitor.In still further embodiments, the protease resistant scaffold and thepeptide comprise a fusion protein.

The present invention also provides cosmetic and/or pharmaceuticalcomposition comprising said at least one peptide that binds to afibroblast growth factor. In some embodiments, the composition iscapable of modulating hair growth. In some preferred embodiments, thecomposition further comprises a scaffold.

The present invention also provides methods for modulating hair growthcomprising: i) providing a composition comprising a peptide containedwithin a scaffold; ii) providing a subject to be treated; and iii)applying the composition to the subject in an area in which hair growthmodulation is desired. In some embodiments, the peptide binds to afibroblast growth factor. In some preferred embodiments, the fibroblastgrowth factor (FGF) is FGF-5. In some particularly preferredembodiments, the scaffold is selected from the group consisting ofBowman-Birk inhibitor, soybean trypsin inhibitor, and Eglin chymotrypsininhibitor. In some alternative embodiments, the peptide is selected fromthe group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 14. In somefurther embodiments, the peptide is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, and13.

The present invention also provides methods for decreasing the activityof a fibroblast growth factor comprising the steps of: i) providing asubject; and ii) administering a composition comprising at least onepeptide that binds FGF to the subject, under conditions such that theactivity of the fibroblast growth factor is decreased. In someembodiments, the fibroblast growth factor is FGF-5.

The present invention also provides compositions comprising an FGF-5peptide sequence selected from the group of SEQ ID NOS:2, 4, 6, 8, 10,12, 14, 15-43, 45-65, 68-84, 86, 88, 90, 92, 24, 26, 98, and 100.

In additional embodiments, the present invention provides applicationsfor hair and/or skin treatment, as well as applications wound healing,treatment of proliferative diseases, etc. Thus, the present inventionprovides compositions and methods suitable for application in/on humansand other animals.

DESCRIPTION OF THE FIGURES

FIG. 1A provides the plasmid map for pME30.16.

FIG. 1B provides the plasmid map for p2JM103-DNNDPI-BBI.

FIG. 1C provides the plasmid map for pCB04.

FIG. 2 provides the first screen data obtained. In this graph, BLAactivity is plotted against total BLA activity. The x-axis representstotal BLA activity and the Y-axis represents bound BLA activity, asdescribed in the Examples.

FIG. 3 provides data from the second screen, with the normalized datasets plotted against each other. The X-axis represents the normalizedbackground, while the Y-axis represents the bound normalized, asdescribed in the Examples.

FIG. 4 provides purification results for BLA-peptide fusion proteinspurified by IMAC chromatography, as described in the Examples.

FIG. 5 provides an SDS-PAGE showing imidazole-eluted BLA activefragments, as described in the Examples.

FIG. 6 provides results of an assay for binding concentrations forpeptide-BLA fusions. Panel A provides results for the followingpeptides: 2E2, 2H10, 2H9 and 1A6 and WT, as described in the Examples.Panel B provides results of an assay for binding concentrations forpeptide-BLA fusions for the following peptides against a negativecontrol: 2E2, 2H10, 2H9 and 1A6 and WT, as described in the Examples.Panel C provides results of an assay for binding concentrations forpurified peptide-BLA fusions for the following peptides: 1C2 and 2E5 andWT. Panel D provides results of an assay for binding concentrations forpurified peptide-BLA fusions for the following peptides against anegative control: 1C2 and 2E5 and WT.

FIG. 7 provides FGF-5 binding curves. Panel A provides a standardbinding curve, as described in the Examples. Various concentrations ofFGF-5 were added to quiescent NR6R-3T3 cells and proliferation wasmeasured as described in the Examples. Panel B provides a binding curveshowing pre-incubation of FGF-5+FGFrI(IIIc) to determine theneutralizing effect of antagonist, a receptor, on FGF-5 activity, andproliferation measured accordingly, as described in the Examples. PanelC provides a binding curve showing pre-incubation of BLA, alone, inaddition to quiescent NR6R-3T3 cells and then measurement ofproliferation, as described in the Examples. Panel D provides bindingcurves given pre-incubation of BLA-peptide fusions+FGF-5, the mixturebeing added to quiescent NR6R-3T3 cells with proliferation measured asdescribed in the Examples. The peptide portions shown in the Figure are2E2, 2H10, 2H9, and 1A6. Panel E provides binding curve data forpre-incubation of BLA-peptide fusions+FGF-5, the mixture being added toquiescent NR6R-3T3 cells with proliferation measured as described in theExamples. The peptide portions shown in the Figure are 1C2 and 2E5.

FIG. 8 provides the amino acid sequence (SEQ ID NO:44) of BBI backbone(71 amino acid residues). The loops are underlined.

FIG. 9 provides the sequence of FGF-5-binding BBI-peptide constructs(SEQ ID NOS:84-101) for mature BCE103 cellulase-BBI fusions with FGF-5binders (See, Table 1) in either the trypsin or chymotrypsin loop, asindicated. Panel A provides the nucleotide and amino acid sequences of aconstruct with the 1A6 peptide in the trypsin loop. Panel B provides thenucleotide and amino acid sequences of a construct with the 1C2 peptidein the trypsin loop. Panel C provides the nucleotide and amino acidsequences of a construct with the 2E2 peptide in the trypsin loop. PanelD provides the nucleotide and amino acid sequences of a construct withthe 2E5 peptide in the trypsin loop. Panel E provides the nucleotide andamino acid sequences of a construct with the 1A6 peptide in thechymotrypsin loop. Panel F provides the nucleotide and amino acidsequences of a construct with the 1C2 peptide in the chymotrypsin loop.Panel G provides the nucleotide and amino acid sequences of a constructwith the 2E2 peptide in the chymotrypsin loop. Panel H provides thenucleotide and amino acid sequences of a construct with the 2E5 peptidein the chymotrypsin loop. The chymotrypsin and trypsin loops areunderlined in each of above Panels.

FIG. 10 provides several selected binders from the screen set forth inExample 4. The left column indicates the clone designation, followed bythe indication of plate, position and readings between the control plateand target plate. The last column provides the sequence of the selectedbinder.

FIG. 11 provides two graphs (Panels A and B) showing results fromBioVeris assays (Example 11) for selected BBI-peptide constructs. Thepercent binding was determined by subtracting the background (wild-typeBBI) and calculating out of 100.

FIG. 12 provides a graph showing cell-based assay (Example 10) resultsobtained for wildtype BBI (WT-BBI) and two BBI-peptide constructs.

DESCRIPTION OF THE INVENTION

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, and recombinant DNA, which are within the skill of theart. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works (See e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual”, Second Edition (ColdSpring Harbor), [1989]); and Ausubel et al., “Current Protocols inMolecular Biology” [1987]). All patents, patent applications, articlesand publications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham,The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used in the invention. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceof the present invention, the preferred methods and materials aredescribed herein. Accordingly, the terms defined immediately below aremore fully described by reference to the Specification as a whole. Also,as used herein, the singular “a,” “an,” and “the” includes the pluralreference unless the context clearly indicates otherwise. Numeric rangesare inclusive of the numbers defining the range. Unless otherwiseindicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

Definitions

As used herein, the term “fibroblast growth factor” (FGF) refers to anymember of this family of proteins, known to those of skill in the art.In particularly preferred embodiments, the term is used in reference toFGF-5.

As used herein, in some embodiments, the “compound” comprises the“complete” protein, (i.e., in its entire length as it occurs in nature(or as mutated)), while in other embodiments it comprises a truncatedform of a protein. Thus, the compounds of the present invention areeither truncated or be “full-length.” In addition, in some embodiments,the truncation is located at the N-terminal end, while in otherembodiments the truncation is located at the C-terminal end of theprotein. In further embodiments, the compound lacks one or more portions(e.g., sub-sequences, signal sequences, domains or moieties), whetheractive or not.

The term “organism” is used throughout the specification to describe ananimal, preferably a human, to whom treatment, including prophylactictreatment, with the compounds according to the present invention isprovided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the term organism refers to that specific animal.

The “host cells” used in the present invention generally are prokaryoticor eukaryotic hosts which contain an expression vector and/or gene ofinterest. Host cells are transformed or transfected with vectorsconstructed using recombinant DNA techniques. Such transformed hostcells are capable of either replicating vectors encoding the proteinvariants or expressing the desired protein variant. In the case ofvectors which encode the pre- or prepro-form of the protein variant,such variants, when expressed, are typically secreted from the host cellinto the host cell medium.

The term “effective amount” is used throughout the specification todescribe concentrations or amounts of compounds according to the presentinvention which may be used to produce a favorable change in the diseaseor condition treated, whether that change is hair growth or preventionof hair growth.

As used herein, “vitamin B₃ compound” means a compound having theformula:

wherein R is —CONH₂ (i.e., niacinamide), —COOH (i.e., nicotinic acid) or—CH₂OH (i.e., nicotinyl alcohol); derivatives thereof; and salts of anyof the foregoing.

As used herein, “non-vasodilating” means that an ester does not commonlyyield a visible flushing response after application to the skin in thesubject compositions. It is contemplated that the majority of thegeneral population would not experience a visible flushing response,although such compounds may cause vasodilation not visible to the nakedeye.

As used herein, “retinoid” includes all natural and/or synthetic analogsof Vitamin A and/or retinol-like compounds which possess the biologicalactivity of Vitamin A in/on the skin, as well as the geometric isomersand stereoisomers of these compounds.

As used herein, the term “bioactivity” refers to a cause and effectrelationship between a composition and a biological system. Thus, theterm is used as by those skilled in the art of biotechnology andbiological sciences as the phrase that describes a cause and effectrelationship between a molecular composition and living biologicalmatter (e.g., tissue, cells, etc.).

As used herein as a noun, the term “bioactive” refers a composition thatexhibits bioactivity upon administration to living biological matter(e.g., tissue, cells, etc.). The term is used synonymously with“bioactive compound.”

As used herein, “silicone gum” means high molecular weight siliconeshaving an average molecular weight in excess of about 200,000 andpreferably from about 200,000 to about 4,000,000. It is intended thatthe definition encompass non-volatile polyalkyl and polyaryl siloxanegums.

As used herein, the term “polypeptide” refers to a compound made up of asingle chain of amino acid residues linked by peptide bonds. The term“protein” herein may be synonymous with the term “polypeptide” or mayrefer, in addition, to a complex of two or more polypeptides. The exactmeaning is that known to those in the art.

As used herein, the terms “expression cassette” and “expression vector”refer to nucleic acid constructs generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a target cell. Therecombinant expression cassette can be incorporated into a plasmid,chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acidfragment. Typically, the recombinant expression cassette portion of anexpression vector includes, among other sequences, a nucleic acidsequence to be transcribed and a promoter. The term “expressioncassette” may be used interchangeably herein with “DNA construct” andits grammatical equivalents.

As used herein, the terms “vector” and “cloning vector” refer to nucleicacid constructs designed to transfer nucleic acid sequences into cells.

As used herein, the term “expression vector” refers to a vector that hasthe ability to incorporate and express heterologous DNA fragments in aforeign cell. Many prokaryotic and eukaryotic expression vectors arecommercially available. Selection of appropriate expression vectors iswithin the knowledge of those of skill in the art.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes orintegrates into the host chromosomes.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of the geneor the chemical synthetic peptide. The process includes bothtranscription and translation of the gene to producepolypeptide/protein.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain that may or may not include regionspreceding or following the coding region.

As used herein, the terms “nucleic acid molecule” and “nucleic acidsequence” include sequences of any form of nucleic acid, including, butnot limited to RNA, DNA and cDNA molecules. It will be understood that,as a result of the degeneracy of the genetic code, a multitude ofnucleotide sequences encoding a given protein may be produced, inaddition to mutant proteins.

As used herein, “codon” refers to a sequence of three nucleotides in aDNA or mRNA molecule that represents the instruction for incorporationof a specific amino acid into a polypeptide chain.

As used herein, the term “disulfide bridge” or “disulfide bond” refersto the bond formed between the sulfur atoms of cysteine residues in apolypeptide or a protein. In this invention, a disulfide bridge ordisulfide bond may be non-naturally occurring and introduced by way ofpoint mutation.

As used herein, the term “salt bridge” refers to the bond formed betweenoppositely charged residues, amino acids in a polypeptide or protein. Inthis invention, a salt bridge may be non-naturally occurring andintroduced by way of point mutation.

As used herein, an “enzyme” refers to a protein or polypeptide thatcatalyzes at least one chemical reaction.

As used herein, the term “activity” refers to any activity associatedwith a particular protein, such as enzymatic activity associated with aprotease. In some embodiments, the activity is biological activity. Infurther embodiments, activity encompasses binding of proteins toreceptors which results in measurable downstream effects, such asinhibition of the transition from anagen to catagen, as describedherein. “Biological activity” refers to any activity that would normallybe attributed to that protein by one skilled in the art.

As used herein, the term “protease” refers to an enzyme that degradespeptide bonds.

As used herein, “peptide bond” refers to the chemical bond between thecarbonyl group of one amino acid and the amino group of another aminoacid.

As used herein, “wild-type” refers to a sequence or a protein that isnative or naturally occurring.

As used herein, “point mutations” refers to a change in a singlenucleotide of DNA, especially where that change results in a sequencechange in a protein.

As used herein, “mutant” refers to a version of an organism or proteinwhere the version is other than wild-type. The change may be effected bymethods well known to one skilled in the art, for example, by pointmutation in which the resulting protein may be referred to as a mutant.

As used herein, “mutagenesis” refers to the process of changing acomposition (e.g., protein) from a wild-type composition (e.g., protein)into a mutant or variant composition (e.g., protein).

As used herein, “substituted” and “substitutions” refer toreplacement(s) of an amino acid residue or nucleic acid base in a parentsequence. In some embodiments, the substitution involves the replacementof a naturally occurring residue or base.

As used herein, “modification” and “modify” refer to any change(s) in anamino acid or nucleic acid sequence, including, but not limited todeletions, insertions, interruptions, and substitutions. In someembodiments, the modification involves the replacement of a naturallyoccurring residue or base.

As used herein, “functional portion of a secreted polypeptide” and itsgrammatical equivalents refers to a truncated secreted polypeptide thatretains its ability to fold into a normal, albeit truncated,configuration. In some embodiments, it is contemplated that sufficientresidues of a domain of the naturally secreted polypeptide must bepresent to allow it to fold in its normal configuration independently ofthe desired polypeptide to which it is attached. However, in most cases,the portion of the secreted polypeptide are both correctly folded andresult in increased secretion as compared to its absence. Similarly, inmost cases, the truncation of the secreted polypeptide means that thefunctional portion retains a biological function. In a preferredembodiment, the catalytic domain of a secreted polypeptide is used,although other functional domains may be used, for example, thesubstrate binding domains. Additionally preferred embodiments utilizethe catalytic domain and all or part of the linker region.

As used herein, “loop” refers to a sequence of amino acids, for example3-20 amino acids, more preferably 5-15 amino acids, even more preferably5-10 amino acids, and most preferably 7-9 amino acids, which connectsstructural elements of a protein. Such elements include, but are notlimited to beta sheets and helical elements and the connecting loop of abeta-hairpin. In some embodiments, the loop is further stabilizedthrough the use of covalent linkages. In some preferred embodiments, thecovalent linkages comprise disulfide bonds, especially as providedherein. In alternative embodiments, the loops are stabilized by the useof other means, including but not limited to amides, hydrogen bonds,and/or salt bridges. In most embodiments, the loops are located on thesurface of proteins and may be altered, as provided herein, to conferadditional (e.g., desirable) properties to the requisite proteins.

As used herein, “oligonucleotide” refers to a short nucleotide sequencewhich may be used, for example, as a primer in a reaction used to createmutant proteins.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequencewhich encodes a gene product. The coding region may be present in eithera cDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labelled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method well-known in the art (See e.g., U.S. Pat. Nos. 4,683,1954,683,202, and 4,965,188, hereby incorporated by reference), forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the term “RT-PCR” refers to the replication andamplification of RNA sequences. In this method, reverse transcription iscoupled to PCR, most often using a one enzyme procedure in which athermostable polymerase is employed, as described in U.S. Pat. No.5,322,770, herein incorporated by reference. In RT-PCR, the RNA templateis converted to cDNA due to the reverse transcriptase activity of thepolymerase, and then amplified using the polymerizing activity of thepolymerase (i.e., as in other PCR methods).

As used herein, the term “hybridization” refers to the process by whicha strand of nucleic acid joins with a complementary strand through basepairing, as known in the art.

As used herein, “maximum stringency” refers to the level ofhybridization that typically occurs at about Tm-5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologs.

The phrases “substantially similar and “substantially identical” in thecontext of two nucleic acids or polypeptides typically means that apolynucleotide or polypeptide comprises a sequence that has at least 75%sequence identity, preferably at least 80%, more preferably at least90%, still more preferably 95%, most preferably 97%, sometimes as muchas 98% and 99% sequence identity, compared to the reference (i.e.,wild-type) sequence. Sequence identity may be determined using knownprograms such as BLAST, ALIGN, and CLUSTAL using standard parameters.(See e.g., Altschul, et al., J. Mol. Biol. 215:403-410 [1990]; Henikoffet al., Proc. Natl. Acad Sci. USA 89:10915 [1989]; Karin et al., Proc.Natl Acad. Sci USA 90:5873 [1993]; and Higgins et al., Gene 73:237-244[1988]). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. Also,databases may be searched using FASTA (Pearson et al., Proc. Natl. Acad.Sci. USA 85:2444-2448 [1988]).

As used herein, “equivalent residues” refers to proteins that shareparticular amino acid residues. For example, equivalent resides may beidentified by determining homology at the level of tertiary structurefor a protein (e.g., FGF-5) whose tertiary structure has been determinedby x-ray crystallography. Equivalent residues are defined as those forwhich the atomic coordinates of two or more of the main chain atoms of aparticular amino acid residue of the protein having putative equivalentresidues and the protein of interest (N on N, CA on CA, C on C and O onO) are within 0.13 nm and preferably 0.1 nm after alignment. Alignmentis achieved after the best model has been oriented and positioned togive the maximum overlap of atomic coordinates of non-hydrogen proteinatoms of the proteins analyzed. The preferred model is thecrystallographic model giving the lowest R factor for experimentaldiffraction data at the highest resolution available, determined usingmethods known to those skilled in the art of crystallography and proteincharacterization/analysis.

In some embodiments, modification is preferably made to the “precursorDNA sequence” which encodes the amino acid sequence of the precursorenzyme, but can be by the manipulation of the precursor protein. In thecase of residues which are not conserved, the replacement of one or moreamino acids is limited to substitutions which produce a variant whichhas an amino acid sequence that does not correspond to one found innature. In the case of conserved residues, such replacements should notresult in a naturally-occurring sequence. Derivatives provided by thepresent invention further include chemical modification(s) that changethe characteristics of the protein.

In some preferred embodiments, the protein gene is ligated into anappropriate expression plasmid. The cloned protein gene is then used totransform or transfect a host cell in order to express the protein gene.In some embodiments, this plasmid replicates in the hosts, in the sensethat it contains the well-known elements necessary for plasmidreplication or the plasmid may be designed to integrate into the hostchromosome. The necessary elements are provided for efficient geneexpression (e.g., a promoter operably linked to the gene of interest).In some embodiments, these necessary elements are supplied as the gene'sown homologous promoter if it is recognized, (i.e., transcribed, by thehost), a transcription terminator (a polyadenylation region foreukaryotic host cells) which is exogenous or is supplied by theendogenous terminator region of the protein gene. In some embodiments, aselection gene such as an antibiotic resistance gene that enablescontinuous cultural maintenance of plasmid-infected host cells by growthin antimicrobial-containing media is also included.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule which is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule which is expressed from arecombinant DNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (i.e., FGF-5 and/or fragmentsthereof) joined to an exogenous protein fragment (the fusion partnerwhich consists of a non-FGF-5 protein). In some embodiments, the fusionpartner enhances solubility of the FGF-5 protein as expressed in a hostcell, may provide an affinity tag to allow purification of therecombinant fusion protein from the host cell or culture supernatant, orboth. If desired, the fusion protein may be removed from the protein ofinterest (i.e., FGF-5 and/or fragments thereof) by a variety ofenzymatic or chemical means known to the art.

The terms “in operable combination,” “in operable order,” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “structural gene” refers to a DNA sequencecoding for RNA or a protein. In contrast, “regulatory genes” arestructural genes which encode products which control the expression ofother genes (e.g., transcription factors).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, recombinant FGF-5polypeptides are expressed in host cells and the polypeptides arepurified by the removal of host cell proteins; the percent ofrecombinant FGF-5 polypeptides is thereby increased in the sample.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNA s which encode a multitude of proteins.However, isolated nucleic acid encoding a FGF-5 protein includes, by wayof example, such nucleic acid in cells ordinarily expressing a FGF-5protein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, “anagen” refers to the active growth phase of hairfollicles. In the anagen phase, cells in the root of the hair dividerapidly, adding to the hair shaft. During this phase, the hair growsabout 1 cm every 28 days. Scalp hair stays in this active phase ofgrowth for 2-6 years.

As used herein, “catagen” refers to the hair growth phase that occurs atthe end of the anagen phase. It signals the end of the active growth ofa hair. This phase lasts for about 2-3 weeks while a club hair isformed.

As used herein, “telogen” refers to the resting phase of the hairfollicle. At any given time, 10%-15% of all hairs are in the telogenphase. This phase lasts for about 100 days for hairs on the scalp andmuch longer for hairs on the eyebrows, eyelashes, arms and legs. Duringthis phase, the hair follicle is completely at rest and the club hair iscompletely formed. Pulling out a hair in this phase will reveal a solid,hard, dry, white material at the root. About 25-100 telogen hairs areshed normally each day.

As used herein, “depilation” refers to the act of removing hair.“Depilatories” are compositions that are used to remove hair.

As used herein, “alopecia” refers to loss of hair. Hair loss is thoughtto proceed by mechanisms involving FGF-5, as described herein. However,it is not intended that the present invention be limited to anyparticular mechanism in hair loss.

As used herein, “cosmetic composition” refers to compositions that finduse in the cosmetics. The Food Drug and Cosmetic Act (FD&C Act)definition is used herein. Thus, cosmetics are defined by their intendeduse, as articles intended to be rubbed, poured, sprinkled, or sprayedon, introduced into, or otherwise applied to the human body forcleansing, beautifying, promoting attractiveness, or alteringappearance. These compositions provide non-therapeutic benefits and arenot regulated as pharmaceuticals. However, in some situations, cosmeticcompositions are incorporated into pharmaceutical compositions toprovide cosmetic benefits (e.g., products that treat skin or hairdiseases, but also contain cosmetic compositions for their coloring orother benefits). Also, it is intended that the present inventionencompass the use of cosmetics on animals other than humans.

As used herein, the terms “pharmaceutical compositions” and “therapeuticcompositions” refer to compositions such as drugs that provide medicalbenefits, rather than solely cosmetic benefits. In the United States,pharmaceutical and therapeutic compositions are approved by the Food andDrug Administration for treatment and/or prevention of particularconditions.

As used herein, the term “drug” is defined as it is in the FD&C Actdefinition. Thus, drugs are defined as articles intended for use in thediagnosis, cure, mitigation, treatment or prevention of disease, andarticles (other than food) intended to affect the structure or anyfunction of the body of man or other animals.

As used herein, the term “cosmetic benefit” refers to a desired cosmeticchange that results from the administration of a personal carecomposition. Cosmetic benefits include but are not limited toimprovements in the condition of skin, hair, nails, and the oral cavity.In preferred embodiments, at least one cosmetic benefit is provided bythe skin care, hair care, nail care, and makeup compositions of thepresent invention.

As used herein, “skin care composition” refers to compositions that areapplied to skin in order to provide beneficial properties, including butnot limited to wrinkle minimizing, wrinkle removal, decoloring,coloring, skin softening, skin smoothing, dipilation, cleansing, etc. Insome particularly preferred embodiments, the present invention providesskin care compositions that improve skin tone. In these embodiments, theimprovement comprises lessening of wrinkles, smoothing skin texture,modifying skin coloration, and other desired cosmetic benefits.

As used herein, “hair care composition” refers to compositions that areapplied to hair to provide beneficial properties such as thickening,thinning, coloring, decoloring, cleansing, conditioning, softening,shaping, etc.

As used herein, “makeup compositions” refer to cosmetic preparationsthat are used to beautify, caring for, maintaining, or augment theappearance of a human or other animal. “Makeup compositions” include,but are not limited to color cosmetics, such as mascaras, lipsticks, lipliners, eye shadows, eye-liners, rouges, face powders, foundations,blushes, and nail polish.

As used herein, the term “dispersed phase” is used as by those of skillin the art of emulsion technology as the phase that exists as smallparticles or droplets suspended in and surrounded by a continuous phase.The dispersed phase is also known as the “internal” or “discontinuous”phase.

As used herein, “penetration enhancers” refer to compositions thatfacilitate penetration through the upper stratum corneum barrier to thedeeper skin layers. Examples of penetration enhancers include, but arenot limited to, propylene glycol, azone, ethoxydiglycol, dimethylisosorbide, urea, ethanol, dimethyl sulfoxide, micoroemulsions,liposomes, and nanoemulsions.

As used herein, the terms “emulsifier” and “surfactant” refer tocompounds that disperse and suspend the dispersed phase within thecontinuous phase of a material. Surfactants find particular use inproducts intended for skin and/or hair cleansing. In particularembodiments, the term “surfactant(s)” is used in reference tosurface-active agents, whether used as emulsifiers or for othersurfactant purposes such as skin cleansing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptides and supported peptides that bindFGF-5.

In some embodiments, the present invention provides cosmetic and/orpharmaceutical compounds for modulating hair growth comprising at leastone polypeptide or a peptide. In some preferred embodiments, thecompounds comprise a polypeptide.

In alternative preferred embodiments, the compounds comprise at leastone peptide. In some preferred embodiments, the peptide has an aminoacid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6,8, 10, 12, and 14 (See, Table 1). In some preferred embodiments, thecompound has a sequence, the sequence being at least 70%, preferably80%, more preferably 90%, and most preferably 95% homologous to thesequences set forth herein. In some preferred embodiments, thepolypeptide has a molecular weight that is preferably between 500Daltons and 30,000 Daltons, more preferably between 1000 Daltons and10,000 Daltons, and most preferably from 1500 Daltons to 8,000 Daltons.

In some preferred embodiments, modulation comprises treatment of atleast one disease or condition that involves loss of hair. In somepreferred embodiments, the disease or condition is at least one selectedfrom the group consisting of inflammatory alopecias, pseudopelade,scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheicdermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia,alopecia areata, cancer, conditions that affect defective hair fiberproduction, and environmental factors that affect hair production. Insome preferred embodiments, the disease is androgenic alopecia oralopecia areata.

In some preferred embodiments, modulation comprises hair growthinhibition and/or hair removal for treatment of at least one disease orcondition for which decreased hair growth is desirable. In somepreferred embodiments, inhibition and/or removal comprises depilation.

In some preferred embodiments, the invention provides cosmetic and/orpharmaceutical compounds for modulating hair growth comprising at leastone peptide or polypeptide and at least one scaffold, the peptide orpolypeptide being contained in the scaffold, preferably the peptide orpolypeptide being a loop, and most preferably, the loop being closed bya disulfide bond. In preferred embodiments, the peptide or polypeptidebinds to FGF-5. In particularly preferred embodiments, the bindingresults in the blocking of the FGF-5 downstream activity. In somepreferred embodiments, the scaffold is STI, Eglin or BBI. Inparticularly preferred embodiments, the scaffold is BBI. In furtherpreferred embodiments, the peptide or polypeptide comprises apolypeptide.

In additional preferred embodiments, the compound comprises at least onepeptide. In some preferred embodiments, the peptide has an amino acidsequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8,10, 12, and 14 (See, Table 1). In other preferred embodiments, thecompounds has an amino acid sequence comprising any sequence selectedfrom the group consisting of SEQ ID NOS: 15-43 (See, FIG. 10). In somepreferred embodiments, the compound has a sequence, the sequence beingat least 70%, more preferably 80%, even more preferably 90%, and mostpreferably 95% homologous to the sequences set forth herein. In apreferred embodiment, the polypeptide has a molecular weight that ispreferably between 500 Daltons and 100,000 Daltons, more preferablybetween 500 Daltons and 30,000 Daltons, even more preferably between 500Daltons and 45,000 Daltons, still more preferably between 1000 Daltonsand 12,000 Daltons, and most preferably from 1500 Daltons to 10,000Daltons.

In some preferred embodiments, modulation comprises treatment of atleast one disease or condition that involves loss of hair. In somepreferred embodiments, the disease or condition is at least one selectedfrom the group consisting of inflammatory alopecias, pseudopelade,scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheicdermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia,alopecia areata, cancer, conditions that affect defective hair fiberproduction, and environmental factors that affect hair production. Insome particularly preferred embodiments, the disease is androgenicalopecia or alopecia areata.

In some preferred embodiments, modulation comprises hair growthinhibition and/or hair removal for treatment of at least one disease orcondition for which decreased hair growth is desirable. In somepreferred embodiments, inhibition and/or removal comprises depilation.

In yet further embodiments, the present invention provides cosmeticand/or pharmaceutical compositions comprising a polypeptide or peptide,as set forth herein, and a physiologically acceptable carrier orexcipient. Preferably, the compound is present in an amount of about0.0001% to about 5% by weight based on the total weight of thecomposition. Also preferably, the compound is present in an amount ofabout 0.001% to about 0.5% by weight based on the total weight of thecomposition. The composition may be in the form of an emulsifiedvehicle, such as a nutrient cream or lotion, a stabilized gel ordispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In yet further embodiments, the present invention provides means fordecreasing FGF-5 activity. In some embodiments, the method comprisingapplying an effective amount of at least one of the compounds describedherein to an organism in need thereof.

In additional preferred embodiments, the present invention is directedto at least one peptide or polypeptide, at least one loop and at leastone protease-resistant scaffold. Flexible native loops are found on thesurface of most protein modules and exist as short stretches of aminoacids that connect regions of defined secondary structure. Althoughcrystallographic and NMR (nuclear magnetic resonance) studies show thatnative loops are usually less well defined than alpha-helices andbeta-sheets, their conformational freedom is normally restrictedsubstantially compared with free peptides. Consequently, the bindingactivities of native loops in proteins usually differ significantly fromthose of the corresponding linear amino acid sequence. However, it isnot intended that the present invention be limited to any specificmechanism.

The loops provided by the present invention bind proteins such as FGF(e.g., FGF-5). Binding the loop to the protein prevents the protein frombinding to its target. Thus, binding interactions are thought to bedisrupted by binding the loop to the protein. As a result, bioactivitycan be altered as desired. However, it is not intended that the presentinvention be limited to any particular mechanism.

The present invention further provides scaffolds to stabilize the loops.STI, BBI and EglinC have native loops that bind to and inhibitproteases. In some embodiments, STI and BBI native loops are replacedwith the polypeptides and/or peptides of the invention. In someembodiments, these sequences are replaced with inhibitors or enhancersof any FGF, while in other embodiments, the sequences are replaced withinhibitors or enhancers of FGF-5. In additional embodiments, STI and BBInative loops are replaced with sequences that have been isolated usingvarious techniques known in the art (e.g., phage display), such as theFGF-5 binding proteins described herein.

In some embodiments, a native loop is replaced with a loop which is 3 to20 amino acids in length, preferably 5 to 15 amino acids in length, andmore preferably 5 to 10 amino acids in length. In alternativeembodiments, longer sequences find use, as long as they provide bindingand/or inhibition. In addition, peptides suitable for use asreplacements of the native loop(s) can form constrained loops (i.e., aloop formed by the presence to a disulfide bond between two cysteineresidues). In some particularly preferred embodiments, the peptides arebetween 7 and 9 amino acids in length.

There are several advantages to using scaffolds to stabilize peptidesequences. In some preferred embodiments, the biological activity of thepeptide is higher and/or effectively modulates biological function as aresult of limiting peptide flexibility and reducing the entropic cost offixing the polypeptide sequence in the bioactive conformation. Inaddition, structural information obtained by x-ray crystallography findsuse in guiding affinity maturation. Furthermore, in some embodiments,the sequence presented on a structural scaffold is more resistant toproteolytic degradation in different biological applications. In stillfurther embodiments, the chimeric construction is obtained in largeamount in low cost biological expression systems for industrialapplications.

As shown in the Examples, in some embodiments, the present inventionprovides compounds that bind FGF-5. In some embodiments, binding absorbsextracellular FGF-5, thereby preventing FGF-5 from interacting with itscognate ligand, when then prevents downstream biological effect(s). Inthe present invention, binding prevents FGF-5 from interacting with itscognate receptor and inhibits transition from the anagen to the catagenpromoting hair growth and preventing hair loss.

BBI represents a class of protein scaffolds whose binding to proteasesis mediated by an exposed native loop that is fixed in a characteristiccanonical conformation and which fits into the active site in a mannerthought to be similar to that of a substrate (Laskowski and Kato, Ann.Rev. Biochem., 49:593-626 [1980]; and Bode & Huber, supra). The nativeloop is frequently constrained by the presence of disulfide bridgesand/or extensive hydrogen-bonding networks that act to lock thestructure into the correct canonical structure. The sequence of thisloop determines the specificity of the inhibition, which mirrors thespecificity of proteases for their substrates. For example, most trypsininhibitors have Arg or Lys as their P1 residue. Inhibitors of the BBIfamily have a network of conserved disulfide bridges that help form asymmetrical structure of two tricyclic domains (Chen et al., supra;Werner and Wemmer, supra; and Liu et al., supra), each containing anindependent serine protease binding site. The native binding loop iscontained within a region joined by disulfide bridges formed betweencysteine residues. The identity of the amino acid residue at the P1 siteon each domain is the main determinant of the serine protease inhibited.Native domains possess lysine or arginine for trypsin, leucine ortyrosine for chymotrypsin and alanine for elastase (Tsunogae et al., J.Biochem. (Tokyo) 100:243-246 [1986]). In addition, serine is highlyconserved at the P′1 position and proline at the P′3 position. Theindividual native loop regions of BBI are well suited for protein loopgrafting of previously identified cysteine constrained peptides thatbind to targets selectively, as described herein.

Numerous isoforms of BBI have been characterized. For example, SEQ IDNO:44 (See, FIG. 8) provides the amino acid sequence of a BBI backbonedescribed herein comprising approximately 71 amino acid residues. Inaddition, in some embodiments BBI is truncated with as many as 10 aminoacid residues being removed from either the N- or C-terminal. Any of theisoforms described herein, as well as those additional ones known in theart, find use as scaffolds in the present invention.

The present invention provides several advantages over creation of, forexample, chimeric proteins. Transfer of an entire protein can bedifficult when, for example, a protein domain of interest carries morethan one important biological activity. Maintaining one activity (e.g.functionally significant domain-domain interactions) while alteringanother (e.g. high affinity binding to a co-factor or receptor) can beproblematic. The present invention, as indicated herein, overcomes thatlimitation, as in preferred embodiments the loops are transferred,instead of entire domains.

In addition, in some embodiments, the compounds of the present inventioncomprise at least one mutation in addition to those set out above. Othermutations, such as deletions, insertions, substitutions, transversions,transitions and inversions, at one or more other locations, also finduse in the present invention.

In some embodiments, the compounds of the present invention alsocomprise a conservative substitution that may occur as a like-for-likesubstitution (e.g., basic for basic, acidic for acidic, polar for polaretc.). In additional embodiments, non-conservative substitutions areprovided (i.e., from one class of residue to another or alternativelyinvolving the inclusion of unnatural amino acids such as ornithine,diaminobutyric acid ornithine, norleucine ornithine, pyriylalanine,thienylalanine, naphthylalanine and phenylglycine).

In some embodiments, the sequences also have deletions, insertionsand/or substitutions of amino acid residues that produce a silent changeand result in a functionally equivalent substance.

In some embodiments, deliberate amino acid substitutions are made on thebasis of similarity in amino acid properties (e.g., polarity, charge,solubility, hydrophobicity, hydrophilicity, and/or the amphipathicnature of the residues) and it is therefore useful to group amino acidstogether in functional groups. Amino acids can be grouped together basedon the properties of their side chain alone. However it is more usefulto include mutation data as well. The sets of amino acids thus derivedare likely to be conserved for structural reasons. These sets can bedescribed in the form of a Venn diagram (See e.g., Livingstone andBarton, Comput. Appl Biosci., 9:745-756 [1993]; and (Taylor, J. Theor.Biol., 119:205-218 [1986]). In some embodiments, conservativesubstitutions are made, for example according to the table below thatdescribes a generally accepted Venn diagram grouping of amino acids. SetSub-set Hydrophobic F W Y H K M I L V A G C Aromatic F W Y H Aliphatic IL V Polar W Y H K R E D C S T N Q Charged H K R E D Positively H K Rcharged Negatively E D charged Small V C A G S P T N D Tiny A G S

In some embodiments, variant amino acid sequences of the presentinvention also include suitable spacer groups inserted between any twoamino acid residues of the sequence including alkyl groups such asmethyl, ethyl or propyl groups in addition to amino acid spacers such asglycine or β-alanine residues. A further form of variation involves thepresence of one or more amino acid residues in peptoid form.

In some embodiments, homology comparisons find use in identifyinghomologous sequences that find use in the present invention. Homologycomparisons can be conducted by eye, or more usually, with the aid ofreadily available sequence comparison programs. Available computerprograms can calculate the percent homology between two or moresequences. Additionally, percent homology may be calculated overcontiguous sequences (i.e., one sequence is aligned with the othersequence and each amino acid in one sequence is directly compared withthe corresponding amino acid in the other sequence one residue at atime). This is called an “ungapped” alignment. Typically, such ungappedalignments are performed only over a relatively short number ofresidues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalizing unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximize local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment, so that for the same number of identicalamino acids, a sequence alignment with as few gaps as possible (i.e.,reflecting higher relatedness between the two compared sequences) willachieve a higher score than one with many gaps. “Affine gap costs” aretypically used that charge a relatively high cost for the existence of agap and a smaller penalty for each subsequent residue in the gap. Thisis one of the most commonly used gap scoring system. High gap penaltieswill of course produce optimized alignments with fewer gaps. Mostalignment programs allow the gap penalties to be modified. However, itis preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (See e.g., Devereux etal., Nuc. Acids Res., 12:387 [1984]). Examples of other softwarepackages than can perform sequence comparisons include, but are notlimited to, the BLAST package FASTA, and the GENEWORKS suite ofcomparison tools, all of which are well-known to those in the art. BothBLAST and FASTA are available for offline and online searching. However,for some applications, it is preferred to use the GCG Bestfit program.The BLAST 2 Sequence package is also available for comparing protein andnucleotide sequences.

Although the final percent homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pair-wisecomparison based on chemical similarity or evolutionary distance. Anexample of such a matrix commonly used is the BLOSUM62 matrix—thedefault matrix for the BLAST suite of programs. GCG Wisconsin programsgenerally use either the public default values or a custom symbolcomparison table if supplied. For some applications, it is preferred touse the public default values for the GCG package, or in the case ofother software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in DNASIS™ (Hitachi Software), based on analgorithm, analogous to CLUSTAL (See e.g., Higgins and Sharp, Gene73:237-244 [1988]).

Once the software has produced an optimal alignment, it is possible tocalculate the percent of homology, and more preferably, the percent ofsequence identity. The software typically does this as part of thesequence comparison and generates a numerical result.

In some embodiments, the present invention provides nucleic acidsencoding any of the compounds described herein, as well as complementsthereof. In additional preferred embodiments, the invention providesvectors comprising a compound, as disclosed herein, cells comprising thecompound and methods of expressing the compound.

Those of skill in the art appreciate the relationship between nucleicacid sequences and polypeptide sequences, in particular as relate to thegenetic code and the degeneracy of this code, and will be able toconstruct such nucleic acids without difficulty. For example, oneskilled in the art is aware that for each amino acid substitution in asequence there may be one or more codons that encode the substituteamino acid. Accordingly, it is evident that, depending on the degeneracyof the genetic code with respect to that particular amino acid residue,one or more nucleic acid sequences may be generated corresponding tothat polypeptide sequence.

Mutations in amino acid sequence and nucleic acid sequence may be madeby any of a number of techniques, as known in the art. In particularlypreferred embodiments, the mutations are introduced into parentsequences by means of PCR (polymerase chain reaction) using appropriateprimers. In some embodiments, the parent enzymes are modified at theamino acid level, while in other embodiments, the enzymes are modifiedat the nucleic acid level, in order to generate the sequences describedherein. In some preferred embodiments, the present invention providesfor the generation of compounds by introducing one or more correspondingcodon changes in the nucleotide sequence encoding a compound. It will beappreciated that the above codon changes will find use in variousnucleic acid sequences of the present invention. For example, in someembodiments, sequence changes are made to any of the homologoussequences described herein.

As indicated above, in some embodiments, the “compound” comprises the“complete” protein, (i.e., in its entire length as it occurs in nature(or as mutated)), while in other embodiments it comprises a truncatedform of a protein. Thus, the compounds of the present invention areeither truncated or be “full-length.” In addition, in some embodiments,the truncation is located at the N-terminal end, while in otherembodiments the truncation is located at the C-terminal end of theprotein. In further embodiments, the compound lacks one or more portions(e.g., sub-sequences, signal sequences, domains or moieties), whetheractive or not.

In yet further alternative embodiments, the nucleotide sequencesencoding the compounds are prepared synthetically by establishedstandard methods (e.g. the phosphoroamidite method described by Beucageet al., Tetrahedr. Lett., 22:1859-1869 [1981]; or the method describedby Matthes et al., EMBO J., 3:801-805 [1984]). In the phosphoroamiditemethod, oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors.

In some embodiments of the present invention, the nucleotide sequencesare either of mixed genomic and synthetic origin, mixed synthetic andcDNA origin or mixed genomic and cDNA origin, prepared by ligatingfragments of synthetic, genomic or cDNA origin, in accordance withstandard techniques. Each ligated fragment corresponds to various partsof the entire nucleotide sequence. In some embodiments, the DNA sequenceis prepared by polymerase chain reaction (PCR) using specific primers,as known in the art.

In some embodiments, the nucleotide sequences described here andsuitable for use in the methods and compositions described here includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude, but are not limited to methylphosphonate and phosphorothioatebackbones and/or the addition of acridine or polylysine chains at the 3′and/or 5′ ends of the molecule. However, it is not intended that thepresent invention be limited to any particular method, as any suitablemethod known to those in the art for modifying nucleotide sequences finduse in the present invention. In some embodiments, these modificationsare performed in order to enhance the in vivo activity and/or life spanof nucleotide sequences.

In some preferred embodiments, the present invention provides nucleotidesequences and methods for using nucleotide sequences that arecomplementary to the sequences presented herein, as well as derivativesand/or fragments of these sequences.

In some embodiments, the polynucleotides of the present invention finduse in the production of primers and/or probes. Thus, in someembodiments, the polynucleotide sequences are used to produce PCRprimers, primers for other amplification methods as known in the art,labeled probes, and/or for cloning methods. In preferred embodiments,these primers, probes and other fragments are at least 15, preferably atleast 20, and in some more preferable embodiments, at least 25, 30 or 40nucleotides. In addition, these primers, probes and fragments areencompassed by the term “polynucleotide.”

In some embodiments, polynucleotides such as DNA polynucleotides andprobes are produced recombinantly, while in other embodiments they areproduced synthetically. In additional embodiments, these sequences arecloned using standard methods. In general, primers are produced bysynthetic means, involving a stepwise manufacture of the desired nucleicacid sequence one nucleotide at a time. Techniques for accomplishingthis using automated techniques are readily available in the art.However, it is not intended that the present invention be limited toproduction of polynucleotides using any particular method, as anysuitable method known to those in the art finds use in the presentinvention.

In some embodiments, longer polynucleotides are generally be producedusing recombinant means, for example using PCR cloning techniques, asknown in the art. In such embodiments, the primers are typicallydesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be readily cloned into a suitable cloningvector. Preferably, the variant sequences are at least as biologicallyactive as the sequences presented herein.

In some preferred embodiments, sequences that are provided that arecomplementary to the compound or sequences that are capable ofhybridizing to the nucleotide sequences of the compounds (includingcomplementary sequences of those presented herein), as well asnucleotide sequences that are complementary to sequences that canhybridize to the nucleotide sequences of the compounds (includingcomplementary sequences of those presented herein). In some preferredembodiments, polynucleotide sequences that are capable of hybridizing tothe nucleotide sequences presented herein under conditions ofintermediate to maximal stringency are provided.

In some preferred embodiments, nucleotide sequences that can hybridizeto the nucleotide sequence of the compound nucleic acid, or thecomplement thereof, under stringent conditions (e.g., 50° C. and0.2×SSC) are provided. More preferably, the nucleotide sequences canhybridize to the nucleotide sequence of the compound, or the complementthereof, under more highly stringent conditions (e.g. 65° C. and0.1×SSC).

In some embodiments, it is desirable to mutate the sequence in order toprepare a compound. Accordingly, in some embodiments, mutants areprepared from the compounds provided herein. In some embodiments,mutations are introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites. Various methods known in the art find use in thisembodiment (See e.g., Morinaga et al., Biotechnol., 2:646-649 [1984];Nelson and Long, Anal. Biochem., 180:147-151 [1989]; and Sarkar andSommer, Biotechn., 8:404-407 [1990]). However, additional methods finduse in the present invention and it is not intended that the presentinvention be limited to any particular method.

In some preferred embodiments, the sequences used in the methods andcompositions described herein is a recombinant sequence (i.e., asequence that has been prepared using recombinant DNA techniquesproduced using any suitable method known in the art.

In additional embodiments, the present invention provides vectorscomprising the compound, cells comprising the compound, and methods ofexpressing the compound. In some embodiments, the nucleotide sequencesused in the methods and compositions described herein are incorporatedinto a recombinant replicable vector. In some embodiments, the vector isused to replicate and express the nucleotide sequence, in enzyme form,in and/or from a compatible host cell. In some embodiments, expressionis controlled using control sequences (e.g., regulatory sequences). Insome embodiments, proteins produced by a host cell by expression of thenucleotide sequence are secreted (i.e., into the growth medium), whilein other embodiments, the proteins are contained intracellularly withinthe host cell. In some embodiments, the coding sequences are designed toinclude signal sequences which direct secretion of the substance codingsequences through a particular prokaryotic or eukaryotic cell membrane.In further embodiments, polynucleotides are incorporated into arecombinant replicable vector. In additional embodiments, the vector isused to replicate the nucleic acid in a compatible host cell. Inpreferred embodiments, the vector comprising the polynucleotide sequenceis transformed into a suitable host cell. While any suitable host findsuse in the present invention, in some preferred embodiments, the hostsare selected from the group consisting of bacterial, yeast, insect,fungal, and mammalian cells.

In some embodiments, compounds and their polynucleotides are expressedby introducing a polynucleotide into a replicable vector, introducingthe vector into a compatible host cell, and growing the host cell underconditions which bring about replication of the vector. In someembodiments, the vector is recovered from the host cell.

In additional embodiments, the compound nucleic acid is operativelylinked to transcriptional and translational regulatory elements activein the host cell. In some embodiments, the compound nucleic acid alsoencodes a fusion protein comprising at least one signal sequence (e.g.,those derived from the glucoamylase gene from Schwanniomycesoccidentalis, α-factor mating type gene from Saccharomyces cerevisiaeand the TAKA-amylase from Aspergillus oryzae). In further alternativeembodiments, the compound nucleic acid encodes a fusion proteincomprising a membrane binding domain.

In some preferred embodiments, the compound is expressed at the desiredlevels in a host organism using an expression vector. It is contemplatedthat any expression vector comprising a compound nucleic acid that iscapable of expressing the gene encoding the compound nucleic acid in theselected host organism will find use in the present invention. Thechoice of vector depends upon the host cell into which it is to beintroduced. Thus, in some embodiments, the vector is an autonomouslyreplicating vector (i.e., a vector that exists as an episomal entity,the replication of which is independent of chromosomal replication, suchas, for example, a plasmid, a bacteriophage or an episomal element, aminichromosome or an artificial chromosome). Alternatively, in someembodiments, the vector integrates into the host cell genome andreplicates together with the chromosome.

In some preferred embodiments, the expression vector includes thecomponents of a cloning vector, including but not limited to suchcomponents as an element that permits autonomous replication of thevector in the selected host organism and one or more phenotypicallydetectable markers for selection purposes. In preferred embodiments, theexpression vector further comprises control nucleotide sequencesencoding a promoter, operator, ribosome binding site, translationinitiation signal, and optionally, a repressor gene or one or moreactivator genes. Additionally, in some embodiments, the expressionvector comprises a sequence coding for an amino acid sequence capable oftargeting the compound to a host cell organelle such as a peroxisome orto a particular host cell compartment. Such a targeting sequenceincludes but is not limited to the sequence SKL. For expression underthe direction of control sequences, the nucleic acid sequence encodingthe compound is operably linked to the control sequences in propermanner with respect to expression.

In some preferred embodiments, the polynucleotide in a vector isoperably linked to a control sequence that is capable of providing forthe expression of the coding sequence by the host cell (i.e., the vectoris an expression vector). In some embodiments, the control sequences aremodified (e.g., by the addition of further transcriptional regulatoryelements) in order to make the level of transcription directed by thecontrol sequences more responsive to transcriptional modulators. In somepreferred embodiments, the control sequences comprise promoters.

In some preferred embodiments of the vectors, the nucleic acid sequenceencoding for the compound is operably combined with a suitable promotersequence. The promoter can be any DNA sequence having transcriptionactivity in the host organism of choice and can be derived from genesthat are homologous or heterologous to the host organism. Examples ofsuitable promoters for directing the transcription of the modifiednucleotide sequence, such as compound nucleic acids, in a bacterial hostinclude, but are not limited to the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, thepromoters of the Bacillus licheniformis α-amylase gene (amyL), the aprEpromoter of Bacillus subtilis, the promoters of the Bacillusstearothermophilus maltogenic amylase gene (amyM), the promoters of theBacillus amyloliquefaciens α-amylase gene (amyQ), the promoters of theBacillus subtilis xylA and xylB genes and a promoter derived from aLactococcus sp.-derived promoter including the P170 promoter. When thegene encoding the compound is expressed in a bacterial species such asE. coli, a suitable promoter can be selected, for example, from abacteriophage promoter including a T7 promoter and a phage lambdapromoter. For transcription in a fungal species, examples of usefulpromoters are those derived from the genes encoding the Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase, and A. nidulans acetamidase. Examplesof suitable promoters for the expression in a yeast species include butare not limited to the Gal 1 and Gal 10 promoters of Saccharomycescerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.

Examples of suitable bacterial host organisms are Gram positive species,including, but not limited to members of the Bacillaceae, (e.g., B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothennophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. lautus, B.megaterium and B. thuringiensis), Streptomyces species (e.g., S. murinusand S. lividans) lactic acid bacteria (e.g., Lactococcus spp. such asLactococcus lactis; Lactobacillus spp. including Lactobacillus reuteri;Leuconostoc spp.; Pediococcus spp.; and Streptococcus spp.Alternatively, strains of Gram-negative species belonging toEnterobacteriaceae (e.g., E. coli) or members of the Pseudomonadaceaefind use in the present invention.

In some embodiments, a suitable yeast host organism is selected fromvarious biotechnologically useful yeasts species, including but notlimited to Pichia sp., Hansenula sp or Kluyveromyces, Yarrowinia,Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyce(e.g., S. pombe). In some embodiments, strains of the methylotrophicyeast species Pichia pastoris are used as the host organism, while inother embodiments, the host organism is a Hansenula species. Suitablehost organisms among filamentous fungi include species of Aspergillus(e.g., A. niger, A. oryzae, A. tubigensis, A. awamori and Aspergillusnidulans). Alternatively, strains of Fusarium species (e.g. F.oxysporum) and Rhizomucor (e.g., Rhizomucor miehei) find used as thehost organism. Additional suitable strains include, but are not limitedto Thermomyces and Mucor species.

In some preferred embodiments, host cells comprising polynucleotides areused to express polypeptides, such as the compounds disclosed herein,fragments, homologues, variants or derivatives thereof. Host cells arecultured under suitable conditions which allow expression of theproteins. In some embodiments, expression of the polypeptides isconstitutive (i.e., the peptides are continually produced), while inother embodiments, expression is inducible. In the case of inducibleexpression, protein production is initiated when required by addition ofan inducer substance to the culture medium (e.g., dexamethasone orIPTG). Polypeptides can be extracted from host cells by a variety oftechniques known in the art, including enzymatic, chemical, and/orosmotic lysis and physical disruption. Indeed, it is not intended thatthe present invention be limited to any particular means of harvestingexpressed polypeptides.

In alternative embodiments, polypeptides are produced recombinantly inany suitable (including commercially available) in vitro cell-freesystem, such as the TnT™ (Promega) rabbit reticulocyte system.

In additional preferred embodiments, the present invention providescosmetic and/or pharmaceutical compositions comprising at least onepolypeptide or peptide, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the compound is present inan amount of about 0.0001% to about 5% by weight, based on the totalweight of the composition. Also preferably, the compound is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup. Preferably, the carrier is at least compound selected from thegroup consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In some liposomal embodiments, the liposomes comprise water and one ormore ingredients capable of forming lipid bilayer vesicles that can holdone or more functional or active ingredient(s). Non-limiting examples ofingredients capable of forming lipid bilayer vesicles include:phospholipids, hydrogenated phosphatidylcholine, lecithin, cholesteroland sphingolipids. Non-limiting examples of functional or activeingredients that can be delivered via liposomes include: vitamins andtheir derivatives, antioxidants, proteins and peptides, keratolyticagents, bioflavinoids, terpenoids, phytochemicals, and extracts ofplant, marine or fermented origin. In a preferred embodiment, thecomposition further comprises a skin care or hair care active. Activeingredients can include any of a wide variety of ingredients such as areknown in the art. (See e.g., McCutcheon's Functional Materials, NorthAmerican and International Editions, (2003), published by MC PublishingCo.). Preferably, such actives include but are not limited toantioxidants, such as tocopheryl and ascorbyl derivatives,bioflavinoids, terpenoids, synthetics and the like, vitamins and vitaminderivatives, hydroxyl- and polyhydroxy acids and their derivatives, suchas AHAs and BHAs and their reaction products, peptides and polypeptidesand their derivatives, such as glycopeptides and lipophilized peptides,heat shock proteins and cytokines, enzymes and enzymes inhibitors andtheir derivatives, such as proteases, MMP inhibitors, catalases, glucoseoxidase and superoxide dismutase, amino acids and their derivatives,bacterial, fungal and yeast fermentation products and their derivatives,including mushrooms, algae and seaweed and their derivatives,phytosterols and plant and plant part extracts and their derivatives andphospholipids and their derivatives, anti-dandruff agents such as zincpyrithione and delivery systems containing them, as provided hereinand/or known in the art.

In some preferred embodiments, the skin care active is selected from thegroup consisting of a Vitamin B3 component, panthenol, Vitamin E,Vitamin E acetate, retinol, retinyl propionate, retinyl palmitate,retinoic acid, Vitamin C, theobromine, alpha-hydroxyacid, famesol,phytrantriol, salicylic acid, palmityl peptapeptide-3 and mixturesthereof. In some preferred embodiments, the Vitamin B3 component isniacinamide. In some embodiments, the compositions provided hereincomprise a skin care active at a level from about 0.0001% to about 20%,preferably from about 0.001% to about 5%, more preferably from about0.01% to about 2%, by weight.

Exemplary derivatives of the foregoing vitamin B₃ compounds includenicotinic acid esters, including non-vasodilating esters of nicotinicacid, nicotinyl amino acids, nicotinyl alcohol esters of carboxylicacids, nicotinic acid N-oxide and niacinamide N-oxide. Suitable estersof nicotinic acid include nicotinic acid esters of C₁-C₂₂, preferablyC₁-C₁₆, more preferably C₁-C₆ alcohols. In these embodiments, thealcohols are suitably straight-chain or branched chain, cyclic oracyclic, saturated or unsaturated (including aromatic), and substitutedor unsubstituted. The esters are preferably non-vasodilating.

Non-vasodilating esters of nicotinic acid include tocopherol nicotinateand inositol hexanicotinate; tocopherol nicotinate are preferred. A morecomplete description of vitamin B₃ compounds is provided in WO 98/22085.Preferred vitamin B₃ compounds include niacinamide and tocopherolnicotinate.

In additional embodiments, the skin care active comprises at least oneretinoid. The retinoid is preferably retinol, retinol esters (e.g.,C₂-C₂₂ alkyl esters of retinol, including retinyl palmitate, retinylacetate, retinyl proprionate), retinal, and/or retinoic acid (includingall-trans retinoic acid and/or 13-cis-retinoic acid), more preferablyretinoids other than retinoic acid. These compounds are well known inthe art and are commercially available from a number of sources (e.g.,Sigma and Boehringer Mannheim). Preferred retinoids include retinol,retinyl palmitate, retinyl acetate, retinyl proprionate, retinal,retinoic acid and combinations thereof. More preferred are retinol,retinoic propionate, retinoic acid and retinyl palmitate. In someembodiments, the retinoid is included as a substantially pure material,while in other embodiments, it is provided as an extract obtained bysuitable physical and/or chemical isolation from natural (e.g., plant)sources. When a retinoid is included in the compositions herein, itpreferably comprises from about 0.005% to about 2%, preferably fromabout 0.01% to about 1% retinoid. Retinol is preferably used in anamount of from about 0.01% to about 0.15%; retinol esters are preferablyused in an amount of from about 0.01% to about 2% (e.g., about 1%).

In some embodiments, the compositions of the present invention comprisesafe and effective amounts of a dermatologically acceptable carrier thatis suitable for topical application to the skin or hair within which theessential materials and optional other materials are incorporated toenable the essential materials and optional components to be deliveredto the skin or hair at an appropriate concentration. Thus, in someembodiments, the carrier acts as a diluent, dispersant, solvent or thelike for the essential components, ensuring that these components can beapplied and distributed evenly over the selected target at anappropriate concentration.

In further embodiments, an effective amount of one or more compoundsdescribed herein is also be included in compositions to be applied tokeratinous materials such as nails and hair, including but not limitedto those useful as hair spray compositions, hair styling compositions,hair shampooing and/or conditioning compositions, compositions appliedfor the purpose of hair growth regulation and compositions applied tothe hair and scalp for the purpose of treating seborrhoea, dermatitisand/or dandruff.

In yet additional embodiments, an effective amount of one or morecompounds described herein is included in compositions suitable fortopical application to the skin or hair. These compositions are providedin any suitable form, including but not limited to creams, lotions,gels, suspensions dispersions, microemulsions, nanodispersions,microspheres, hydrogels, emulsions (e.g., oil-in-water and water-in-oil,as well as multiple emulsions), and multilaminar gels and the like (Seee.g., Schlossman et al., The Chemistry and Manufacture of Cosmetics,[1998], incorporated by reference, herein). In some embodiments, thecompositions are formulated as aqueous or silicone compositions, whilein other embodiments they are formulated as emulsions of one or more oilphases in an aqueous continuous phase (or an aqueous phase in an oilphase).

The type of carrier utilized in the present invention depends on thetype of product form desired for the composition. The carrier can besolid, semi-solid or liquid. Suitable carriers include liquids,semi-solids (e.g., creams, lotions, gels, sticks, ointments, andpastes), sprays and mousses. Preferably the carrier is in the form of alotion, cream or a gel, more preferably one which has a sufficientthickness or yield point to prevent the particles from sedimenting. Insome embodiments, the carrier is inert, while in other embodiments itprovides dermatological benefits. In some embodiments, the carrier isapplied directly to the skin and/or hair, while in other embodiments, itis applied via a woven or non-woven wipe or cloth. In yet otherembodiments, it is in the form of a patch, mask or wrap. In stillfurther embodiments, it is aerosolized or otherwise sprayed or pumpedonto the skin and/or hair. The carrier chosen is physically andchemically compatible with the essential components described herein,and should not unduly impair stability, efficacy or other use benefitsassociated with the compositions of the present invention.

Preferred carriers contain a dermatologically acceptable, hydrophilicdiluent. Suitable hydrophilic diluents include water, organichydrophilic diluents such as C₂-C₁₀, preferably C₂-C₆, preferably, C₃-C₆monohydric alcohols and low molecular weight glycols and polyols,including propylene glycol, polyethylene glycol polypropylene glycol,glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters,1,2,6-hexametriol, pentylene glycol, hexylene glycol, sorbitol esters,ethoxylated ethers, propoxylated ethers, and combinations thereof. Thediluent is preferably liquid. Water is a preferred diluent. Thecomposition preferably comprises at least about 20% of the hydrophilicdiluent.

In some embodiments, suitable carriers also comprise an emulsioncomprising a hydrophilic phase, especially an aqueous phase, and ahydrophobic phase (e.g., a lipid, oil or oily material). As well knownto those skilled in the art, the hydrophilic phase is dispersed in thehydrophobic phase, or vice versa, to form respectively hydrophilic orhydrophobic dispersed and continuous phases, depending on thecomposition of ingredients. The term “dispersed phase” is a termwell-known to one skilled in the art of emulsion technology, used inreference to the phase which exists as small particles or droplets thatare suspended in and surrounded by a continuous phase. The dispersedphase is also known as the internal or discontinuous phase. The emulsionmay be or comprise (e.g., in a triple or other multi-phase emulsion) anoil-in-water emulsion or a water-in-oil emulsion such as awater-in-silicone emulsion. Oil-in-water emulsions typically comprisefrom about 1% to about 60% (preferably about 1% to about 30%) of thedispersed hydrophobic phase and from about 1% to about 99% (preferablyfrom about 10% to about 90%) of the continuous hydrophilic phase, whilewater-in-oil emulsions typically comprise from about 1% to about 98%(preferably from about 40% to about 90%) of the dispersed hydrophilicphase and from about 1% to about 50% (preferably about 1% to about 30%)of the continuous hydrophobic phase.

In further embodiments, the carrier also includes one or more componentsthat facilitate penetration through the upper stratum corneum barrier tothe lower levels of the skin. Examples of penetration enhancers include,but are not limited to, propylene glycol, azone, ethoxydiglycol,dimethyl isosorbide, urea, ethanol and dimethyl sulfoxide, as well asmicroemulsions, liposomes and nanoemulsions.

In some additional embodiments, the compositions of the presentinvention comprise humectants which are preferably present at a level offrom about 0.01% to about 20%, preferably from about 0.1% to about 15%and preferably from about 0.5% to about 10%. Preferred humectantsinclude, but are not limited to, compounds selected from polyhydricalcohols, sorbitol, glycerol, urea, betaine, D-panthenol, DL-panthenol,calcium pantothenate, royal jelly, panthetine, pantotheine, panthenylethyl ether, pangamic acid, pyridoxin, pantoyl lactose Vitamin Bcomplex, sodium pyrrolidone carboxylic acid, hexane-1, 2, 6,-triol,guanidine or its derivatives, and mixtures thereof.

Suitable polyhydric alcohols for use herein include, but are not limitedto polyalkylene glycols and preferably alkylene polyols and theirderivatives, including propylene glycol, dipropylene glycol,polypropylene glycol, polyethylene glycol and derivatives thereof,sorbitol, hydroxypropyl sorbitol, erythritol, threitol, pentaerythritol,xylitol, glucitol, mannitol, pentylene glycol, hexylene glycol, butyleneglycol (e.g., 1,3-butylene glycol), hexane triol (e.g.,1,2,6-hexanetriol), trimethylol propane, neopentyl glycol, glycerine,ethoxylated glycerine, propane-1,3 diol, propoxylated glycerine andmixtures thereof. The alkoxylated derivatives of any of the abovepolyhydric alcohols are also suitable for use herein. Preferredpolyhydric alcohols of the present invention are selected fromglycerine, butylene glycol, propylene glycol, pentylene glycol, hexyleneglycol, dipropylene glycol, polyethylene glycol, hexane triol,ethoxylated glycerine and propoxylated glycerine and mixtures thereof.

Suitable humectants useful herein are sodium 2-pyrrolidone-5-carboxylate(NaPCA), guanidine; glycolic acid and glycolate salts (e.g., ammoniumand quaternary alkyl ammonium); lactic acid and lactate salts (e.g.,ammonium and quaternary alkyl ammonium); aloe vera in any of its varietyof forms (e.g., aloe vera gel); hyaluronic acid and derivatives thereof(e.g., salt derivatives such as sodium hyaluronate); lactamidemonoethanolamine; acetamide monoethanolamine; urea; betaine, panthenoland derivatives thereof; and mixtures thereof.

In some embodiments, at least part (up to about 5% by weight ofcomposition) of a humectant is incorporated into the compositions of thepresent invention in the form of an admixture with a particulatecross-linked hydrophobic acrylate or methacrylate copolymer, itselfpreferably present in an amount of from about 0.1% to about 10%, whichcan be added either to the aqueous or disperse phase. This copolymer isparticularly valuable for reducing shine and controlling oil whilehelping to provide effective moisturization benefits and is described infurther detail in WO96/03964, incorporated herein by reference.

In some embodiments, the oil-in-water and water-in-oil compositions ofthe present invention comprise from about 0.05% to about 20%, preferablyfrom about 1% to about 15%, preferably from about 2% to about 10%,preferably from about 2% to about 5% of a dermatologically acceptableemollient. Emollients tend to lubricate the skin, increase thesmoothness and suppleness of the skin, prevent or relieve dryness of theskin and/or protect the skin. Emollients are typically water-immiscible,oily or waxy materials and emollients can confer aesthetic properties toa topical composition. A wide variety of suitable emollients are known(See e.g., Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol.1, pp. 32-43 [1972]; and WO 00/24372), and find use herein, containsnumerous examples of materials suitable as emollients. Additionalemollients include, but are not limited to the following:

i) Straight and branched chain hydrocarbons having from about 7 to about40 carbon atoms, such as mineral oils, dodecane, squalane, cholesterol,hydrogenated polyisobutylene, isohexadecane, isoeicosane,isooctahexacontane, isohexapentacontahectane, and the C₇-C₄₀isoparaffins, which are C₇-C₄₀ branched hydrocarbons. Suitable branchedchain hydrocarbons for use herein are selected fromisopentacontaoctactane, petrolatum and mixtures thereof;

ii) C₁-C₃₀ fatty acid esters of C₁-C₃₀ carboxylic acids, C₁₂₋₁₅ alkylbenzoates and of C₂-C₃₀ dicarboxylic acids, e.g. isononyl isononanoate,isostearyl neopentanoate, isodecyl octanoate, isodecyl isononanoate,tridecyl isononanoate, myristyl octanoate, octyl pelargonate, octylisononanoate, myristyl myristate, myristyl neopentanoate, myristyloctanoate, isopropyl myristate, myristyl propionate, isopropyl stearate,isopropyl isostearate, methyl isostearate, behenyl behenate, dioctylmaleate, diisopropyl adipate, and diisopropyl dilinoleate and mixturesthereof also find use in the present invention;

iii) C₁-C₃₀ mono- and poly-esters of sugars and related materials. Theseesters are derived from a sugar or polyol moiety and one or morecarboxylic acid moieties. Depending on the constituent acid and sugar,these esters can be in either liquid or solid form at room temperature.Examples include: glucose tetraoleate, the galactose tetraesters ofoleic acid, the sorbitol tetraoleate, sucrose tetraoleate, sucrosepentaoleate, sucrose hexaoleate, sucrose heptaoleate, sucroseoctaoleate, sorbitol hexaester. Other materials include cottonseed oilor soybean oil fatty acid esters of sucrose. Other examples of suchmaterials are described in WO 96/16636, incorporated by referenceherein;

iv) Vegetable oils and hydrogenated vegetable oils. Examples ofvegetable oils and hydrogenated vegetable oils include safflower oil,grapeseed oil, coconut oil, cottonseed oil, menhaden oil, palm kerneloil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, ricebran oil, pine oil, nut oil, sesame oil, sunflower seed oil, partiallyand fully hydrogenated oils from the foregoing sources and mixturesthereof;

v) Soluble or colloidally-soluble moisturizing agents. Examples includehyaluronic acid and chondroitin sulfate.

In some embodiments, the compositions of the present invention containan emulsifier and/or surfactant, generally to help disperse and suspendthe disperse phase within the continuous aqueous phase. A surfactant mayalso be useful if the product is intended for skin or hair cleansing.For convenience hereinafter, “emulsifiers” are encompassed by the term“surfactants.” Thus, as used herein, the term “surfactant(s)” refers tosurface active agents, whether used as emulsifiers or for othersurfactant purposes such as skin cleansing. Known, includingconventional surfactants find use in the present invention, providedthat the selected agent is chemically and physically compatible withessential components of the composition and provides the desiredcharacteristics (See e.g., WO 00/24372). Suitable surfactants includenon-silicone derived materials, silicone-derived materials, and mixturesthereof.

In further embodiments, the compositions of the present inventioncomprise preferably from about 0.05% to about 30%, more preferably fromabout 0.5% to 15%, and most preferably from about 1% to 10% of asurfactant or mixture of surfactants. The exact surfactant or surfactantmixture chosen depends upon the pH of the composition, the othercomponents present and the desired final product aesthetics.

Among the nonionic surfactants that are useful herein are those that canbe broadly defined as condensation products of long chain alcohols(e.g., C₈₋₃₀ alcohols), with sugar or starch polymers (e.g.,glycosides). Other useful nonionic surfactants include the condensationproducts of alkylene oxides with fatty acids (i.e., alkylene oxideesters of fatty acids). These materials have the general formulaRCO(X)_(n)OH wherein R is a C₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— (i.e.,derived from ethylene glycol or oxide) or —OCH₂CHCH₃— (i.e., derivedfrom propylene glycol or oxide) and n is an integer from about 6 toabout 200. Other nonionic surfactants are the condensation products ofalkylene oxides with 2 moles of fatty acids (i.e., alkylene oxidediesters of fatty acids). These materials have the general formulaRCO(X)nOOCR wherein R is a C₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— ( i.e.,derived from ethylene glycol or oxide) or —OCH₂CHCH₃—(i.e., derived frompropylene glycol or oxide) and n is an integer from about 6 to about100. In some embodiments, an emulsifier for use herein is preferably afatty acid ester blend based on a mixture of sorbitan fatty acid esterand sucrose fatty acid ester, especially a blend of sorbitan stearateand sucrose cocoate. Further suitable examples include a mixture ofcetearyl alcohols and cetearyl glucosides. However, it is not intendedthat the present invention be limited to any particular emulsifier, asvarious suitable emulsifiers are known in the art.

In additional embodiments, the hydrophilic surfactants useful hereinalternatively or additionally include any of a wide variety of cationic,anionic, zwitterionic, and amphoteric surfactants such as are known inthe art (See, e.g., McCutcheon's, Emulsifiers and Detergents, NorthAmerican and International Editions, MC Publishing Co. [2003]; U.S. Pat.No. 5,011,681 U.S. Pat. No. 4,421,769; and U.S. Pat. No. 3,755,560).

A variety of anionic surfactants are also useful herein (See e.g., U.S.Pat. No. 3,929,678). Exemplary anionic surfactants include, but are notlimited to alkoyl isethionates (e.g., C₁₂-C₃₀), alkyl and alkyl ethersulfates and salts thereof, alkyl and alkyl ether phosphates and saltsthereof, alkyl methyl taurates (e.g., C₁₂-C₃₀), and soaps (e.g.,substituted alkylamine and alkali metal salts, e.g., sodium or potassiumsalts) of fatty acids.

Amphoteric and zwitterionic surfactants are also useful herein. Examplesof preferred amphoteric and zwitterionic surfactants which find use inthe compositions of the present invention are those which are broadlydescribed as derivatives of aliphatic secondary and tertiary amines inwhich the aliphatic radical can be straight or branched chain andwherein one of the aliphatic substituents contains from about 8 to about22 carbon atoms (preferably C₈-C₁₈) and one contains an anionic watersolubilizing group (e.g., carboxy, sulfonate, sulfate, phosphate, orphosphonate). Examples, include but are not limited to alkyl iminoacetates and iminodialkanoates and aminoalkanoates, imidazolinium andammonium derivatives. Other suitable amphoteric and zwitterionicsurfactants are those selected from the group consisting of betaines,sultaines, hydro-xysultaines, and branched and unbranched alkanoylsarcosinates, and mixtures thereof.

In further embodiments, some emulsions of the present invention includea silicone containing emulsifier or surfactant. A wide variety ofsilicone emulsifiers find use herein. These silicone emulsifiers aretypically organically modified organopolysiloxanes, also known to thoseskilled in the art as silicone surfactants. Useful silicone emulsifiersinclude, but are not limited to dimethicone copolyols. These materialsare polydimethyl siloxanes which have been modified to include polyetherside chains such as polyethylene oxide chains, polypropylene oxidechains, mixtures of these chains and polyether chains containingmoieties derived from both ethylene oxide and propylene oxide. Otherexamples include alkyl-modified dimethicone copolyols (i.e., compoundswhich contain C₂-C₃₀ pendant side chains). Still other usefuldimethicone copolyols include materials having various cationic,anionic, amphoteric, and zwitterionic pendant moieties.

In some embodiments, the compositions of the present invention compriseat least one polymeric thickening agent. The polymeric thickening agentsuseful herein preferably have a number average molecular weight ofgreater than about 20,000, more preferably greater than about 50,000,and most preferably greater than about 100,000. In some embodiments, thecompositions of the present invention comprise from about 0.01% to about10%, preferably from about 0.1% to about 8% and more preferably fromabout 0.2% to about 5% by weight of the composition of the polymericthickening agent or mixtures thereof.

Preferred polymer thickening agents for use herein include, but are notlimited to non-ionic thickening agents and anionic thickening agents ormixtures thereof. Suitable non-ionic thickening agents include, but arenot limited to polyacrylamide polymers, crosslinkedpoly(N-vinylpyrrolidones), polysaccharides, natural or synthetic gums,polyvinylpyrrolidone and polyvinylalcohol. Suitable anionic thickeningagents include, but are not limited to acrylic acid/ethyl acrylatecopolymers, carboxyvinyl polymers and crosslinked copolymers of alkylvinyl ethers and maleic anhydride. Commercially available thickeners(e.g., Carbopol; Noveon) find use in some embodiments of the presentinvention. Suitable Carbopol resins may be hydrophobically modified, andother suitable resins are described in WO98/22085, or mixtures thereof.

In some embodiments, the present compositions comprise at least onesilicone oil phase. Silicone oil phase(s) generally comprises from about0.1% to about 20%, preferably from about 0.5% to about 10%, and morepreferably from about 0.5% to about 5%, of the composition. The siliconeoil phase preferably comprises one or more silicone components.

In some embodiments, silicone components are fluids, including straightchain, branched and cyclic silicones. Suitable silicone fluids usefulherein include silicones inclusive of polyalkyl siloxane fluids,polyaryl siloxane fluids, cyclic and linear polyalkylsiloxanes,polyalkoxylated silicones, amino and quaternary ammonium modifiedsilicones, polyalkylaryl siloxanes or a polyether siloxane copolymer andmixtures thereof. Volatile, as well as non-volatile silicone fluids finduse herein. Silicone fluids generally have an average molecular weightof less than about 200,000. In preferred embodiments, suitable siliconefluids have a molecular weight of about 100,000 or less, preferablyabout 50,000 or less, and more preferably about 10,000 or less.Preferably the silicone fluid is selected from silicone fluids having aweight average molecular weight in the range from about 100 to about50,000 and preferably from about 200 to about 40,000. Typically,silicone fluids have a viscosity ranging from about 0.65 to about600,000 mm²s⁻¹, preferably from about 0.65 to about 10,000 mm².s⁻¹ at25° C. The viscosity can be measured by means of a glass capillaryviscometer as set forth in Dow Coming Corporate Test Method CTM0004,Jul. 29, 1970. Suitable polydimethyl siloxanes that can be used hereininclude commercially available compounds (e.g., from the GeneralElectric Company and Dow Corning). Also useful are essentiallynon-volatile polyalkylarylsiloxanes, for example,polymethyl-phenylsiloxanes, having viscosities of about 0.65 to 30,000mm²s⁻¹ at 25° C. (General Electric Company or from Dow Coming). Cyclicpolydimethylsiloxanes suitable for use herein are those having a ringstructure incorporating from about 3 to about 7 (CH₃)₂SiO moieties,preferably about 5 or more.

In additional embodiments, silicone gums find use herein. In somepreferred embodiments, a silicone oil phase comprises a silicone gum ora mixture of silicones including the silicone gum. Typically, siliconegums have a viscosity at 25° C. in excess of about 1,000,000 mm²s⁻¹. Thesilicone gums include dimethicones as known in the art (See e.g., U.S.Pat. No. 4,152,416; and Noll, Chemistry and Technology of Silicones,Academic Press, New York [1968]). Silicone gums such as those describedin General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE54 and SE 76, also find use in the present invention. Specific examplesof silicone gums include polydimethylsiloxane,(polydimethylsiloxane)(methylvinylsiloxane) copolymer,poly(dimethylsiloxane)(diphenyl)(methylvinylsiloxane) copolymer andmixtures thereof. Preferred silicone gums for use herein are siliconegums having a molecular weight of from about 200,000 to about 4,000,000selected from dimethiconol, dimethicone copolyol, dimethicone andmixtures thereof.

In some embodiments, a silicone phase herein preferably comprises asilicone gum incorporated into the composition as part of a siliconegum-fluid blend. When the silicone gum is incorporated as part of asilicone gum-fluid blend, the silicone gum preferably constitutes fromabout 5% to about 40%, especially from about 10% to 20% by weight of thesilicone gum-fluid blend. Suitable silicone gum-fluid blends herein aremixtures consisting essentially of:

(i) a silicone having a molecular weight of from about 200,000 to about4,000,000 selected from dimethiconol, fluorosilicone and dimethicone andmixtures thereof; and

(ii) a carrier which is a silicone fluid, the carrier having a viscosityfrom about 0.65 mm²s⁻¹ to about 100 mm²s⁻¹,

wherein the ratio of i) to ii) is from about 10:90 to about 20:80 andwherein said silicone gum-based component has a final viscosity of fromabout 100 mm²s⁻¹ to about 100,000 mm²s⁻¹, preferably from 500 mm²s⁻¹ toabout 10,000 mm²s⁻¹.

Further silicone components suitable for use in a silicone oil phaseherein include crosslinked polyorganosiloxane polymers, optionallydispersed in a fluid carrier. In general, when present the crosslinkedpolyorganosiloxane polymers, together with its carrier (if present)comprises from about 0.1% to about 20%, preferably from about 0.5% toabout 10%, and more preferably from about 0.5% to about 5% of thecomposition. Such polymers comprise polyorganosiloxane polymerscrosslinked by a crosslinking agent (See e.g., WO98/22085). Examples ofsuitable polyorganosiloxane polymers for use herein include, but are notlimited to methyl vinyl dimethicone, methyl vinyl diphenyl dimethiconeand methyl vinyl phenyl methyl diphenyl dimethicone.

Another class of silicone components suitable for use in a silicone oilphase herein includes polydiorganosiloxane-polyoxyalkylene copolymerscontaining at least one polydiorganosiloxane segment and at least onepolyoxyalkylene segment (See e.g., WO98/22085). Suitablepolydiorganosiloxane-polyalkylene copolymers are available commerciallyunder the tradenames BELSIL® from Wacker-Chemie GmbH. A particularlypreferred copolymer fluid blend for use herein includes Dow CorningDC3225C which has the CTFA designation Dimethicone/Dimethicone copolyol.

In further embodiments, compositions of the present invention comprisean organic sunscreen. In some embodiments, suitable sunscreens have UVAabsorbing properties, while others have UVB absorbing properties, andstill others comprise a mixture thereof. The exact amount of thesunscreen active varies, depending upon the desired Sun ProtectionFactor (i.e., the “SPF”) of the composition, as well as the desiredlevel of UV protection. SPF is a commonly used measure ofphotoprotection of a sunscreen against erythema. The SPF is defined as aratio of the ultraviolet energy required to produce minimal erythema onprotected skin to that required to produce the same minimal erythema onunprotected skin in the same individual. Amounts of the sunscreen usedare preferably from about 2% to about 20%, and more preferably fromabout 4% to about 14%. Suitable sunscreens include, but are not limitedto those approved for use in the United States, Japan, Europe andAustralia. The compositions of the present invention preferably comprisean SPF of about 2 to about 30, preferably about 4 about 30, and morepreferably about 4 to about 15.

In some embodiments, the compositions of the present invention may oneor more UVA absorbing sunscreen actives that absorb UV radiation havinga wavelength of from about 320 nm to about 400 nm. Suitable UVAabsorbing sunscreen actives include, but are not limited todibenzoylmethane (See e.g., Lowe and Shaath (eds.), Sunscreens:Development, Evaluation, and Regulatory Aspects, Marcel Dekker, Inc.)derivatives, anthranilate derivatives such as methylanthranilate andhomomethyl, 1-N-acetylanthranilate, and mixtures thereof. The UVAabsorbing sunscreen active is preferably present in an amount sufficientto provide broad spectrum UVA protection either independently, or incombination with, other UV protective actives which may be present inthe composition.

Suitable UVA sunscreen actives include dibenzoylmethane sunscreenactives and their derivatives. They include, but are not limited to,those selected from 2-methyldibenzoylmethane, 4-methyldibenzoylmethane,4-isopropyldibenzoylmethane, 4-tert-butyldibenzoylmethane, 2,4-dimethyldibenzoylmethane, 2, 5-dimethyldibenzoylmethane, 4,4′-diisopropylbenzoylmethane, 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane,2-methyl-5-isopropyl-4′-methoxydibenzoylmethane,2-methyl-5-tert-butyl4′-methoxy-dibenzoylmethane, 2,4-dimethyl-4′-methoxydibenzoylmethane, 2,6-dimethyl-4′-tert-butyl-4′methoxydibenzoylmethane, and mixturesthereof. Preferred dibenzoyl sunscreen actives include those selectedfrom 4-(1, 1-dimethylethyl)-4′-methoxydibenzoylmethane,4-isopropyldibenzoylmethane, and mixtures thereof. A preferred sunscreenactive is 4-(1, 1-dimethylethyl)-4′-methoxydibenzoylmethane.

The sunscreen active 4-(1, 1-dimethylethyl)-4′-methoxydibenzoylmethane,which is also known as butyl methoxydibenzoylmethane or “avobenzone,” iscommercially available under the names of Parsol® 1789 from GivaudanRoure (International) S. A., and Eusolex® 9020 from Merck & Co., Inc.The sunscreen 4-isoproplydibenzoylmethane, which is also known asisopropyldibenzoylmethane, is commercially available from Merck underthe name of Eusolex® 8020.

In some embodiments, the compositions of the present invention furtherinclude one or more UVB sunscreen actives that absorb(s) UV radiationhaving a wavelength of about 290 nm to about 320 nm. The compositionscomprise an amount of the UVB sunscreen active that is safe andeffective in providing UVB protection either independently, or incombination with, other UV protective actives which may be present inthe compositions. The compositions comprise from about 0.1% to about20%, preferably from about 0.1% to about 12%, and more preferably fromabout 0.5% to about 8% by weight, of each UVB absorbing organicsunscreen, or as mandated by the relevant regulatory authority(s).

A variety of UVB sunscreen actives are suitable for use herein (Seee.g., U.S. Pat No. 5,087,372; U.S. Pat. No. 5,073,371; U.S. Pat. No.5,073,372; U.S. Pat. No. 4,937,370; and U.S. Pat. No. 4,999,186).Preferred UVB sunscreen actives are selected from2-ethylhexyl-2-cyano-3, 2-ethylhexyl N,N-dimethyl-p-aminobenzoate,p-aminobenzoic acid, oxybenzone, homomenthyl salicylate, octylsalicylate, 4,4′-methoxy-t-butyldibenzoylmethane, 4-isopropyldibenzoylmethane, 3-benzylidene camphor, 3-(4-methylbenzylidene)camphor, 3-diphenylacrylate, 2-phenyl-benzimidazole-5-sulphonic acid(PBSA), cinnamate esters and their derivatives such as2-ethylhexyl-p-methoxycinnamate, salicylate esters and their derivativessuch as triethanolamine salicylate, ethylhexyl salicylate, octyldimethylpara-aminobenzoic acid, camphor derivatives and their derivatives, andmixtures thereof. Preferred organic sunscreen actives include2-ethylhexyl-2-cyano-3, 3-diphenylacrylate,2-phenyl-benzimidazole-5-sulphonic acid (PBSA),octyl-p-methoxycinnamate, and mixtures thereof. Salt and acidneutralized forms of the acidic sunscreens are also useful herein.

In some embodiments, at least one agent is added to any of thecompositions useful in the present invention to stabilize the UVAsunscreen to prevent it from photo-degrading on exposure to UV radiationand thereby maintaining its UVA protection efficacy. A wide range ofcompounds are reported to have these stabilizing properties and shouldbe chosen to complement both the UVA sunscreen and the composition as awhole (See e.g., U.S. Pat. Nos. 5,972,316; 5,968,485; 5,935,556;5,827,508; and WO 00/06110). Preferred examples of stabilizing agentsfor use in the present invention include 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, ethyl-2-cyano-3, 3-diphenylacrylate, 2-ethylhexyl-3,3-diphenylacrylate, ethyl-3, 3-bis(4-methoxyphenyl)acrylate,diethylhexyl 2,6 napthalate and mixtures thereof (Symrise ChemicalCompany).

In some embodiments, at least one agent is added to any of thecompositions useful in the present invention to improve the skinsubstantivity of those compositions, particularly to enhance theirresistance to being washed off by water or rubbed off. Examples include,but are not limited to, acrylates/C₁₂₋₂₂ alkylmethacrylate copolymer,acrylate/acrylate copolymer, dimethicone, dimethiconol, graft-copoly(dimethylsiloxane/i-butyl methacrylate), lauryl dimethicone,PVP/Hexadecane copolymer, PVP/Eicosene copolymer, tricontanyl PVP andtrimethoxysiloxysilicate.

In addition to organic sunscreens, in some embodiments, the compositionsof the present invention additionally comprise inorganic physicalsunblocks (See e.g., TFA International Cosmetic Ingredient Dictionary,6^(th) Edition, pp. 1026-28 and 1103 [1995]; Sayre et al., J. Soc.Cosmet. Chem., 41:103-109 [1990]; and Lowe et al., supra). Preferredinorganic physical sunblocks include zinc oxide and titanium dioxide andmixtures thereof.

When used in preferred embodiments, the physical sunblocks are presentin an amount such that the present compositions are transparent on theskin (i.e., non-whitening), preferably from about 0.5% to about 20%,preferably from about 0.5% to about 10%, and more preferably from about0.5% to 5% by weight. When titanium dioxide is used, it can have ananatase, rutile or amorphous structure. Manufacturers of micronizedgrade titanium dioxide and zinc oxide for sunscreen use include, but arenot limited to Tayca Corporation, Uniqema, Shinetsu ChemicalCorporation, Kerr-McGee, Nanophase, Nanosource, Sachtleben, Elementis,and BASF Corporation, as well as their distribution agents and thosecompanies that further process the material for sunscreen use. Physicalsunblock particles (e.g., titanium dioxide and zinc oxide) can beuncoated or coated with a variety of materials including but not limitedto amino acids, aluminum compounds such as alumina, aluminum stearate,aluminum laurate, and the like; carboxylic acids and their salts (e.g.,stearic acid and its salts); phospholipids, such as lecithin; organicsilicone compounds; inorganic silicone compounds such as silica andsilicates and mixtures thereof.

In some preferred embodiments, the composition of the present inventionalso include preservatives. Such preservatives include, but are notlimited to pentylene glycol, ethylene diamine tetra acetate (EDTA) andits salts, chlorhexidine (and its diacetate, dihydrochloride,digluconate derivatives), 1,1,1-trichloro-2-methyl-2-propanol,parachloro metaxylenol, polyhexamethylenebiguanide hydrochloride,dehydroacetic acid, diazolidinyl urea, 2,4-dichlorobenzyl alcohol,4,4-dimethyl-1,3-oxazolidine, formaldehyde, glutaraldehyde,dimethylidantoin, imidazolidinyl urea,5-Chloro-2-methyl-4-isothiazolin-3-one, ortho-phenylphenol,4-hydroxybenzoic acid (“paraben”) and its methyl-, ethyl-, propyl-,isopropyl-, butyl-, isobutyl-esters, trichlosan, 2-phenoxyethanol,phenyl mercuric acetate, quaternium-15, salycilate, salicylic acid andits saltssorbic acid and its salts, iodopropanyl butylcarbamate zincpyrithione, benzyl alcohol, 5-bromo-5nitro-1,3-dioxane,2-bromo-2-nitropropane-1,3-diol, benzoic acid and its salts, sulfites,bisulfites, and benzalkonium chloride.

A variety of optional ingredients such as neutralizing agents, perfumesand perfume solubilizing agents, and coloring agents, also find use insome of the compositions herein. It is preferred that any additionalingredients enhance the skin softness/smoothness benefits of theproduct. In addition it is preferred that any such ingredients do notnegatively impact the aesthetic properties of the product.

Neutralizing agents suitable for use in neutralizing acidic groupcontaining hydrophilic gelling agents herein include sodium hydroxide,potassium hydroxide, ammonium hydroxide, monoethanolamine,diethanolamine, amino methyl propanol, tris-buffer and triethanolamine.

Other optional materials that find use in the present invention includeany of the numerous functional and/or active ingredients known to thoseskilled in the art (See e.g., McCutcheon's Functional Materials, NorthAmerican and International Editions, MC Publishing Co. [2003])Non-limiting examples include keratolytic agents; soluble orcolloidally-soluble moisturizing agents such as hyaluronic acid andchondroitin sulfate; vitamins such as vitamin A, vitamin C, vitamin E,vitamin K and derivatives thereof and building blocks thereof;phytantriol; fatty alcohols such as dodecatrienol; alpha and betahydroxyacids; aloe vera; sphingosines and phytosphingosines,cholesterol; skin whitening agents; N-acetyl cysteine; coloring agents;Examples of alpha hydroxy acids include glycolic acid, lactic acid,malic acid, and citric acid (whether derived synthetically or fromnatural sources and whether used alone or in combination) and theiresters or relevant buffered combinations. Other examples ofalpha-hydroxy acids include: alpha-hydroxy ethanoic acid,alpha-hydroxyoctanoic acid, alpha-hydroxycaprylic acid, andhydroxycaprylic acid. Preferred examples of alpha hydroxy acids includeglycolic acid and lactic acid. It is preferred that alpha hydroxy acidsare used in levels of up to about 10%.

Optional materials include pigments that, where water-insoluble,contribute to and are included in the total level of oil phaseingredients. Pigments suitable for use in the compositions of thepresent invention can be organic and/or inorganic. Also included withinthe term “pigment” are materials having a low color or luster, such asmatte finishing agents, light scattering agents, and formulation aidssuch as micas, seracites, and carbonate salts. Further examples ofsuitable pigments include titanium dioxide, iron oxides, zinc oxide,bismuth oxychloride (whether pre-dispersed and/or pre-coated or not) D&Cdyes and lakes, FD&C colors, natural color additives such as carmine,and mixtures thereof. Depending upon the type of composition, a mixtureof pigments is usually used in preferred embodiments of the presentinvention. Preferred pigments for use herein from the viewpoint ofmoisturization, skin feel, skin appearance and emulsion compatibilityare treated pigments. In some embodiments, the pigments are treated withcompounds, including but not limited to amino acids, silicones, lecithinand ester oils.

In preferred embodiments, the pH of the compositions herein is in therange from about 3.5 to about 10, preferably from about 4 to about 8,and more preferably from about 5 to about 7, wherein the pH of the finalcomposition is adjusted by addition of acidic, basic or buffer salts asnecessary, depending upon the composition of the forms and thepH-requirements of the compounds.

The compositions of the present invention are prepared by standardtechniques well known to those skilled in the art. In general theaqueous phase and/or the oil phase are prepared separately, withmaterials of similar phase partitioning being added in any order. If thefinal product is an emulsion, the two phases are then combined withvigorous stirring and/or homogenization as necessary, to reduce the sizeof the internal phase droplets. Any ingredients in the formulation withhigh volatility, or which are susceptible to hydrolysis or decompositionat high temperatures, are added with gentle stirring towards the end ofthe process, post emulsification if applicable. Dosage frequency andamount will depend upon the desired performance criteria.

In some embodiments of the present invention, methods of decreasingFGF-5 activity are provided. In these embodiments, the methods compriseapplying to an organism in need thereof an effective amount of any oneof the compounds set forth herein. These embodiments also includeapplications drawn to hair treatment, as well as other applications(e.g., wound healing, treatment of proliferative diseases, etc). Inadditional preferred embodiments, the present invention providescompounds for treatment of an organism in need thereof.

In additional embodiments, the present invention provides applicationsfor hair and./or skin treatment, as well as applications wound healing,treatment of proliferative diseases, etc. Thus, the present inventionprovides compositions and methods suitable for application in/on humansand other animals.

Experimental

The following Examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: PI (proteinase inhibitor), BBI (Bowman-Birkinhibitor), STI (Soybean Trypsin inhibitor); ppm (parts per million); M(molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles);mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg(milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL(milliliters); μl and μL (microliters); cm (centimeters); mm(millimeters); μm (micrometers); nm (nanometers); U (units); V (volts);MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s)(hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND(not done); NA (not applicable); rpm (revolutions per minute); H₂O(water); dH₂O (deionized water); (HCI (hydrochloric acid); aa (aminoacid); bp (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copyor complimentary DNA); DNA (deoxyribonucleic acid); ssDNA (singlestranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl(sodium chloride); w/v (weight to volume); v/v (volume to volume); g(gravity); OD (optical density); A₄₀₅ (absorbance at 405 nm); Vmax (themaximum initial velocity of an enzyme catalyzed reaction); FGFrI(IIIc)(FGF-5 receptor); Dulbecco's phosphate buffered solution (DPBS); SOC (2%Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl);Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol, 2.31 g/lKH₂PO₄, and 12.54 g/l K₂HPO₄); OD₂₈₀ (optical density at 280 nm); OD₆₀₀(optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis);PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphatebuffer, pH 7.2]); PBST (PBS+0.25% Tween® 20); PEG (polyethylene glycol);PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); SDS(sodium dodecyl sulfate); bME, BME and βME (beta-mercaptoethanol or2-mercaptoethanol); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine(N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino)ethane-sulfonic acid); TAPS(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS(3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide);DTT (1,4-dithio-DL-threitol); Glut and GSH (reduced glutathione); GSSG(oxidized glutathione); TCEP (Tris[2-carboxyethyl] phosphine); Tris(tris(hydroxymethyl)aminomethane); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); SDS (sodium dodecylsulfate); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Ci (Curies) mCi(milliCuries); μCi (microCuries); TLC (thin layer achromatography); Ts(tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me (methyl);Klenow (DNA polymerase I large (Klenow) fragment); rpm (revolutions perminute); EGTA (ethylene glycol-bis(β-aminoethyl ether) N, N, N′,N′-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla(β-lactamase or ampicillin-resistance gene); PDS (plasma-derived bovineserum that has been dialyzed to remove growht factors; dialysis ofdefibrinated bovine plasma is performed against DMEM for about 6 hoursat 4° C., with stirring, the media is changed and dialysis is continuedovernight; the dialyzed PDS is collected after 24 hours, and sterilefiltered twice through a 0.2 μm filter); FCS and FBS (fetal calf serum);GE Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom);DNA2.0 (DNA2.0, Menlo Park, Calif.); OXOID (Oxoid, Basingstoke,Hampshire, UK); Megazyme (Megazyme International Ireland Ltd., BrayBusiness Park, Bray, Co., Wicklow, Ireland); Corning (Corning LifeSciences, Corning, N.Y.); (NEN (NEN Life Science Products, Boston,Mass.); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo,Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, Tex.); ATCC (AmericanType Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL, GrandIsland, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia(Pharmacia Biotech, Pisacataway, N.J.); NCBI (National Center forBiotechnology Information); Applied Biosystems (Applied Biosystems,Foster City, Calif.); Clontech (CLONTECH Laboratories, Palo Alto,Calif.); Difco (Difco Laboratories, Detroit, Mich.); Oxoid (Oxoid Inc.,Ogdensburg, N.Y.); GIBCO BRL or Gibco BRL (Life Technologies, Inc.,Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad(Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego,Calif.); NEB (New England Biolabs, Beverly, Mass.); Sigma (SigmaChemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford,Ill.); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffinann-La Roche,Basel, Switzerland); EM Science (EM Science, Gibbstown, N.J.); Qiagen(Qiagen, Inc., Valencia, Calif.); Biodesign (Biodesign Intl., Saco,Ma.); Aptagen (Aptagen, Inc., Herndon, Va.); Molecular Devices(Molecular Devices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems,Minneapolis, Minn.); Stratagene (Stratagene Cloning Systems, La Jolla,Calif.); Marsh (Marsh Biosciences, Rochester, N.Y.); Bio-Tek (Bio-TekInstruments, Winooski, Vt.); (Biacore (Biacore, Inc., Piscataway, N.J.);PeproTech (PeproTech, Rocky Hill, N.J.); SynPep (SynPep, Dublin,Calif.); and Microsoft (Microsoft, Inc., Redmond, Wash.).

The FGF-5 binding peptides produced during the development of thepresent invention, some of which were used in the following Examples areprovided below. In this Table, results for various assays are provided.In this Table, “Synthesized” refers to the peptide tested alone, “BLA”refers to BLA-peptide fusions, and “BBI/BCE” refers to BBI/BCE-peptidefusions; “l1” refers to loop 1, and “l2” refers to loop 2; “BV” refersto the BioVeris Assay (Example 11), and “CBA” refers to the cell-basedassays (Example 10). Table of Peptides Synthesized BLA BBI/BCE NameSEQUENCE SEQ ID NO: BV CBA BV CBA BV CBA 1A1 CYGLPFTRC SEQ ID NO:4 1A6CEEIWTMLC SEQ ID NO:8 − + + (12) + + (11), 1C2 CWALTVKTC SEQ ID NO:6 −+/− (12) 2E2 CLTVLWTTC SEQ ID NO:14 − + (12) 2E5 CTLWNRSPC SEQ ID NO:12− + − + + (11, 12) − 2H9 CHYLLTNYC SEQ ID NO:10 − 2H10 CRIHLAHKC SEQ IDNO:2 − FGFns2 TNIDSTP SEQ ID NO:45 FGFh12 HLQTTET SEQ ID NO:46 MM001SLNNLTV SEQ ID NO:47 + + MM003 TNIDSTP SEQ ID NO:48 + + MM004 TNIDSTPSEQ ID NO:49 MM005 LRILANK SEQ ID NO:50 MM007 RTQPYPL SEQ ID NO:51 + ++ + − MM009 LLTPTHN SEQ ID NO:52 + + MM010 ALPTHSN SEQ ID NO:53 + +MM012 TNIDSTP SEQ ID NO:54 MM013 LCRRFEN SEQ ID NO:55 MM014 TNIDSTP SEQID NO:56 MM015 TNIDSTP SEQ ID NO:57 MM016 HLQTTET SEQ ID NO:58 + MM017PLGLCPP SEQ ID NO:59 + + MM018 GYFIPSI SEQ ID NO:60 + MM019 TKIDSTP SEQID NO:61 + MM021 HLQTTET SEQ ID NO:62 + PS1 WNIDSTP SEQ ID NO:63 PS2TWIDSTP SEQ ID NO:64 + + + + − PSB TWIDWTP SEQ ID NO:65

EXAMPLE 1 Dermatological Composition

In this Example, various dermatological compositions comprising any ofthe compounds of the present invention are provided as follows.MOISTURIZING BODYWASH (pH 7) RAW MATERIAL (INCI Designation) AmountDeionized Water QS Glycerin 4.0 PEG-6 Caprylic/Capric Glycerides 4.0Palm Kernel Fatty acids 3.0 Sodium Laureth-3 Sulphate 45.0 Cocamide MEA3.0 Sodium Lauroamphoacetate 25.0 Soybean Oil 10.0 Polyquaternium-100.70 Preservative, fragrance, color QS Compound 1000 ppm

BODY WASH RAW MATERIAL pH 8 pH 6.5 pH 7 (INCI Designation) Amount AmountAmount Deionized water QS QS QS Sodium Laureth Sulphate 12 15 8Cocamidopropyl Betaine 8 10 15 Decyl Glucoside 0 2 1 Polyquaternium-100.25 0 0 Polyquaternium-7 0 0 0.7 Preservative, fragrance, color QS QSQS Compound 250 ppm 500 ppm 1000 ppm

BODY LOTION RAW MATERIAL pH 7 pH 7 pH 7.5 pH 7 (INCI Designation) AmountAmount Amount Amount Deionized Water QS QS QS QS Glycerine 8 8 0 12Isohexadecane 3 3 3 6 Niacinamide 0 3 5 6 Isopropyl Isostearate 3 3 3 3Polyacrylamide (and) 3 3 3 3 Isoparaffin (and) Laureth-7 Petrolatum 4 44 2 Nylon 12 2 2 2.5 2.5 Dimethicone 2 2 2.5 2.5 Sucrose Polycottonseed1.5 1.5 1.5 1.5 Oil Stearyl Alcohol 97% 1 1 1 1 D Panthenol 1 1 1 1DL-alpha Tocopherol 1 1 1 1 Acetate Cetyl Alcohol 95% 0.5 0.5 0.5 1Behenyl Alcohol 1 1 1 0.5 Cetearyl Alcohol (and) 0.4 0.4 0.5 0.5Cetearyl Glucoside Stearic Acid 0.15 0.15 0.15 0.15 PEG-100-Stearate0.15 0.15 0.15 0.15 Preservative, QS QS QS QS fragrance, color Compounds250 ppm 500 ppm 750 ppm 1000 ppm

ULTRA-HIGH MOISTURIZING EMULSION RAW MATERIAL pH 7 pH 7 (INCIDesignation) Amount Amount Deionized water QS QS Glycerin 12 5 PEG 400 010 Niacinamide 5 7 Isohexadecane 5 5 Dimethicone 3 2 Polyacrylamide(and) Isoparaffin (and) 3 3 Laureth-7 Isopropyl Isostearate 2 2Polymethylsilsesquioxane 2 2 Cetyl Alcohol 95% 1 1 Sucrosepolycottonseed oil 1 1 D-Panthenol 1 1 Tocopherol Acetate 1 1 StearylAlcohol 95% 0.5 0.5 Cetearyl Glucoside 0.5 0.5 Titanium dioxide 0.3 0.3Stearic Acid 0.15 0.15 PEG-100-Stearate 0.15 0.15 Preservative,fragrance, color QS QS Compound 250 ppm 100 ppm

MOISTURIZING CREAM RAW MATERIAL pH 7 pH 7 pH 7.5 (INCI Designation)Amount Amount Amount Deionized water QS QS QS Glycerine 3 5 10Petrolatum 3 3 0 Cetyl Alcohol 95% 1.5 1.5 1 Dimethicone Copolyol 2 2 2Isopropyl Palmitate 1 1 0.5 Carbopol 954 (Noveon) 0.7 0.7 0.7Dimethicone (350 cs) 1 1 1 Stearyl Alcohol 97% 0.5 0.5 1 Stearic acid0.1 0.1 0.1 Peg-lOO-stearate 0.1 0.1 0.1 Titanium Dioxide 0.3 0.3 0.3Preservative, color, fragrance QS QS QS Compound 50 ppm 250 ppm 1000 ppm

LEAVE-ON HAIR CONDITIONER RAW MATERIAL (INCI Designation) AmountDeionized Water QS Isostearamidopropyl Morpholine Lactate 6.0Hydroxyethylcellulose 1.0 Preservative, fragrance, color QS Compound1000 ppm

CREAM RINSE (pH 4) RAW MATERIAL (INCI Designation) Amount DeionizedWater QS Behentrimonium Chloride 2.0 Trilaureth-4 Phosphate 1.5 Cetylalcohol 2.0 Citric acid QS Preservative, fragrance, color QS Compound1000 ppm

NOURISHING HAIR CONDITIONER/TREATMENT (pH 6) RAW MATERIAL (INCIDesignation) Amount Deionized Water QS Behentrimonium Methosulfate (and)Cetyl Alcohol 4.0 Wheat germ oil 1.0 Cetyl alcohol 0.5 Propylene glycol5.0 PEG-60 Lanolin 1.0 Panthenol 2.0 Lupin amino acids 1.0 CocodimoniumHydroxypropyl Hydrolyzed Wheat Protein 1.0 Fragrance, preservative,color QS Compound 1000 ppm

CONDITIONING SHAMPOO RAW MATERIAL (INCI Designation) Amount DeionizedWater QS Sodium Laureth Sulfate 30% 27.0 Cocamidopropyl Betaine 3.7Coco-Glucoside (and) Glyceryl Oleate 5.0 Coco-Glucoside (and) GlycolDistearate (and) Glycerine 3.0 Guar Hydroxypropyl Trimonium Chloride 0.1Laureth-2 1.55 Fragrance, preservative, color QS Compound 1000 ppm

ANTI-DANDRUFF SHAMPOO RAW MATERIAL (INCI Designation) Amount DeionizedWater QS Magnesium Aluminum Silicate 1.0 Hydroxypropyl Methylcellulose0.8 Sodium Olefin Sulfate 40% 35.0 Lauramide DEA 4.0 Soyamide DEA 1.0Quaternium-70 Hydrolyzed Collagen 2.0 Zinc Pyrithione 40% 4.0 Fragrance,preservative, color QS Compound 1000 ppm

EXAMPLE 2 Panning of a Phage Displayed Peptide Library

In this Example, experiments conducted involving panning of aphage-displayed peptide library are described. A commercially availablephage peptide library PhD C7C (NEB) was panned against FGF-5, accordingto the manufacturer's instructions. Phage that was still bound to thetarget after extensive washes, including an acid wash, was used as atemplate for PCR reactions.

EXAMPLE 3 Construction of a Peptide-BLA Library for FGF-5

In this Example, construction of a peptide-BLA library is described. PCRproduct after one round of phage panning of FGF-5 was cloned into pME30.16 to obtain library pGV04-L. pGV04-L encodes 9-amino acid peptidesequences fused to the N-terminus of E. cloaceae β-lactamase (BLA) witha pIII signal sequence and C-terminal 6×His tag (See, FIG. 1). Theplasmid also carries a chloramphenicol resistance gene (CAT) as aselectable marker and expression is driven by a lac promoter (Plac). Tomake the inserts the PCR product was used as a template for a PCRreaction using primers with BbsI tails. Oligos: ME 190f: (SEQ ID NO:66)GCTATTCAATGTCAGACGAAGACGTCGTTCCTTTCTATTCTCACTCT ME 190r: (SEQ ID NO:67)GGTGGAGGTTCGGCGTCTTCCCGACTGAATGGCTAT

The cut vector, and stuffer insert (200 bp) were ligated overnight at16° C. in a 1:5 molar ratio respectively, using 10 μl of the DNA mix and10 ul of Takara solution I ligase. Ligations were purified using ZymoResearch DNA clean kit and eluted in 2× μl of water. Then, 5 μl ofligation mix was transformed into 40 μl Top 10 (E. coli)electrocompetent cells (Invitrogen), 260 μl SOC was added and the cellswere grown for 1 h at 37° C. The transformation mix was diluted 1/10 andplated on both LA+5 ppm CMP and LA+5 ppm CMP+0.1 ppm CTX plates,followed by incubation overnight at 37° C.

EXAMPLE 4 Primary Screen of FGF-5-Binding BLA-Peptides Using Nitrocefin

In this Example, experiments conducted for primary screening ofFGF-5-binding peptides are described. Peptides fused to the N-terminusof β-lactamase (BLA) were screened for binding to FGF-5, using enzymaticactivity on the lactamase substrate nitrocefin as the reporter.

Growth and Assay Procedure

Peptide-BLA fusions were expressed in E. coli. The growth medium usedwas Luria Broth plus 5 mg/mL chloramphenicol (CMP). One hundred ml ofthe growth medium was added to each well of a sterile Costar 359896-well plate (Corning). One clonal colony was picked into each wellusing a sterile toothpick, and the plate was covered and placed in ahumidified shaker/incubator for 40 hours at 37° C. After incubation, 100mL “B-PER in Phosphate Buffer” (Pierce) was added to each well, and theplate was gently shaken for 30 minutes at room temperature. Theresulting cell lysate was diluted 10× in PBS. Then, 100 ml per well ofthe diluted cell lysate was added to the assay plate (assay platepreparation is described below), which was then covered with a platesealer (Marsh) and incubated for two hours at room temperature. Theassay plate was transferred to a 96-well plate washer (Bio-Tek), wherethe diluted cell lysate was aspirated and the plate washed once with 100ml per well, then 3 times with 300 mL PBS plus 0.1% Tween-20 (PBS-T) perwell. Then, 200 mL nitrocefin working solution was immediately added toeach well. Nitrocefin working solution was prepared just prior to use,by diluting a 100 mg/mil (in DMSO) stock 1000-fold in PBS plus 0.125%n-Octyl-Beta-D-Glucopyranoside (Sigma). After addition of the nitrocefinworking solution, the plate was shaken at room temperature for oneminute, then read for 5 minutes at 495 nm on a Spectramax platespectraphotometer (Molecular Devices) in kinetic mode. Total BLAactivity in the cell lysate was also be measured by diluting cell lysate1:100, then combining 20 ml per well of this dilution with 180 ml perwell of the nitrocefin working solution.

Assay Plate Preparation

A Costar 3594 assay plate (Corning) was prepared by incubating 100 mLper well of a 1 mg/mL solution of FGF-5 (R&D Systems) in 50 mM sodiumcarbonate, pH 9.6. The plate was covered with a plate sealer (Marsh) andincubated two hours at room temperature. The plate was transferred to a96-well plate washer (Bio-Tek), where the FGF-5 solution was aspiratedand the plate washed 3 times with 300 ml PBS-T per well. “Blocker Caseinin PBS” (Pierce) was added at 300 mL per well and the plate wasincubated overnight at 4 degrees C. “Blocker Casein in PBS” wasaspirated and the plate washed 3 times with 300 ml PBS-T per well. Theplate was used in the assay immediately following the last wash. Forscreens in which it was important to distinguish specific FGF-5 bindersfrom nonspecific (background) binders, a no-target control plate wasprepared, beginning with the addition of “Blocker Casein in PBS”, andfollowing the same steps as described.

Data Analysis

In the primary screen, throughput was high priority, and the no-targetcontrol plate was left out of the procedure. The BLA activity of thebound material was plotted against the total BLA activity in the sample,and points that lie above a y-axis cutoff were selected as winners (See,FIG. 2). The cutoff was arbitrarily set by the researcher at a valuewhich, for that specific data set, seems likely to separate true bindersfrom the general mass of data points (See, FIG. 2).

In the secondary screen, triplicate samples of the “winners” from theprimary screen were grown and assayed together, and a no-target controlcondition was run in parallel, to distinguish FGF-5 binders fromnonspecific (background) binders. Wild-type BLA were grown and assayed,to give a measure of the natural “stickiness” of the BLA portion of thefusion molecule. The data were normalized for expression by dividing theFGF-5-binding and background-binding activity by the total BLA activityfor every well, and the normalized data sets were plotted against eachother. Points that lie significantly closer to the “Normalized Bound”axis than the bulk of the data field were selected as winners.Significance was determined by a visual assessment of each point'slocation in the data field, and the size of its error bars. The slope ofthe data set deviated slightly from unity, indicating either nonspecificstickiness due to the presence of FGF-5 or plate-to-plate variation fromthe assay. The results of these experiments are provided in FIG. 3.

EXAMPLE 5 Secondary Screen of FGF-5-Binding-BLA-Peptides UsingNitrocefin

This Example describes secondary screening methods used to analyze FGF-5binding npeptides. Individual clones were picked and grown up in 5 mlLB+5 ppm CMP overnight at 37° C. The cell pastes were then treated with125μ of B-PER reagent (Pierce) for 30 min with slow mixing. COSTARplates (96-well) were coated with 0.1 μg (100 μl of 1 μg/ml) FGF-5,carrier-free (R&D Systems #237-F5/CF, Lot #GQ18304A) with gentle rockingat 4° C. overnight, followed by blocking with Superblock blocking buffer(Pierce) for several hours at room temperature. Then, 100 μl B-Pertreated supernatant from individual clones from pGV02-L were added tothe FGF-5 coated plates. After one hour, plates were washed six timeswith PBS, 0.05% TWEEN®-20 and 200 μl of nitrocefin assay buffercontaining 0.1 mg/ml nitrocefin (Oxoid) was added to measure residualbound beta-lactamase activity, using Abs₄₉/min. Control wells containedpCB04 (See, FIG. 1C) beta-lactamase as a control. Out of 180 clones, 7showed a positive signal. These clones were sequenced, and the resultsare provided in Table 1, where the first column is the name of thepeptide, the second column is the nucleotide sequence and the thirdcolumn is the amino acid sequence of the peptide: TABLE 1 ClonesSequenced Amino Acid Name Nucleotide Sequence Sequence 2H10TGTCGGATTCATCTGGCGCATAAGTGC (SEQ ID NO:1) CRIHLAHKC (SEQ ID NO:2) 1A1TGTTATGGGCTGCCTTTTACGCGGTGC (SEQ ID NO:3) CYGLPFTRC (SEQ ID NO:4) 1C2TGTTGGGCGCTTACTGTGAAGACTTGC (SEQ ID NO:5) CWALTVKTC (SEQ ID NO:6) 1A6TGTGAGGAGATTTGGACTATGCTGTGC (SEQ ID NO:7) CEEIWTMLC (SEQ ID NO:8) 2H9TGTCATTTATCTGCTGACTAATTATGC (SEQ ID NO:9) CHYLLTNYC (SEQ ID NO:10) 2E5TGTACTCTTTGGAATCGTTCTCCGTGC (SEQ ID NO:11) CTLWNRSPC (SEQ ID NO:12) 2E2TGTCTGACGGTGTTGTGGACGACTTGC (SEQ ID NO:13) CLTVLWTTC (SEQ ID NO:14)

EXAMPLE 6 Purification of Sequenced Clones

In this Example, experiments involving purification of the BLA-peptidefusion proteins of the three clones in Example 5 are described. Thesefusion proteins were expressed in E. coli (TOP10; Invitrogen) in 15-mlshake flasks in the presence of 5 ppm CMP and 0.1 ppm cefotoxime (CTX)antibiotic at 37° C. overnight. A commercially available purificationkit (Insect RoboPop Ni/NTA His Bind Purification Kit, Novagen) was usedto purify these peptide BLA fusions according to the manufacturer'sinstructions.

EXAMPLE 7 Peptide-BLA Screen Using Purified Clones

Cell pastes were harvested from the 200 ml cell cultures described inExample 6, by centrifugation at 3,000×g for 10 min. The pastes were thentreated with 25 ml of B-PER reagent (Pierce) for 40 min with slowmixing. The extract was separated by centrifugation at 20,000 g for 20min. BLA activity of all liquid fractions was assayed using nitrocefin,and the concentration of fusion proteins in each fraction was calculatedassuming the same specific activity as the WT enzyme. Fusion proteinswere purified by IMAC chromatography (See, FIG. 4). The imidazole-elutedBLA-active fractions were pooled and the purity was found better than95% as checked by SDS-PAGE. (See, FIG. 5).

The binding (BLA assay) was repeated using different peptideconcentrations to see a dose response. To verify specific binding, theassay was also done using an irrelevant target. The results are shown inFIG. 6 and in Table 2, below. TABLE 2 BLA Binding Assay Results SamplePercent Bound at 0.1 nM 2E2 7 2H10 8 2H9 5 1A6 8 WT (wild-type) 0.4

EXAMPLE 8 BIAcore™ Binding Analysis of FGF-5 Binding BLA-Peptides

In this Example, experiments conducted to determine the affinities ofthe peptides for FGF-5 are described. Affinities of the peptides forFGF-5 were measured using a BIAcore™-3000 surface plasmon resonancesystem (Biacore). A CM5 sensor chip was conditioned with 50 mM NaOH,0.1% HCl, 0.1% SDS and 0.08% H₃PO₄ and activated for covalent couplingof FGF-5 using N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions (Biacore). Human FGF-5 (PeproTech) was dilutedto 5 μg/mL in 20 mM sodium acetate, pH 4.8, and injected at a flow rateof 2 μL/min to achieve approximately 1000 to 2000 response units (RU) ofcoupled protein. An additional solution of EDC and NHS was injected toimprove baseline stability and a solution of 1 M ethanolamine wasinjected as a blocking agent. The reference lane was activated with EDCand NHS and blocked with ethanolamine.

Peptides were synthesized using standard FMOC chemistry, purified byreverse phase HPLC to >95% purity (SynPep) and stored at 10 mg/mL ineither water or 10% DMSO. For kinetic measurements, three-fold orfour-fold serially diluted peptides in HBS-EP buffer, 0.01 M HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20 (Biacore), wereinjected at 25° C. at a flow rate of 50 μL/min. Serially diluted DMSOsamples and buffer samples were also injected for backgroundmeasurements and subtraction. Two short injections of 25 mM NaOH wereused between sample injections for regeneration. Kinetic parameters werecalculated using software programs, Scrubber version 1.1f, BIAevaluation3.1 and Clamp99 version 3.30.

EXAMPLE 9 Construction of FGF-5Binding-BBI-Peptide Constructs

In this Example, methods used in the construction of FGF-5-bindingBBI-peptide constructs are described. The FGF5 binding peptides wereintroduced into the BBI trypsin inhibitory loop or chymotrypsininhibitory loop using the QuikChange® II XL Site-Directed MutagenesisKit (Stratagene). The mutagenesis reaction was performed essentially asdescribed in the technical manual provided by the manufacturer(Stratagene). The vector, p2JM103-DNNDI-BBI (See, FIG. 1), was used as atemplate. This B. subtilis expression vector has the BCE103 cellulasefused to BBI with a cleavable linker between the two domains. In themutagenesis reaction, approximately 200 ng of the plasmid was used with50 pmol of each of the oligonucleotide primers (shown below). Thereaction mixture was held at 97° C. for 3 minutes and then held at 50°C. until the polymerase was added to each tube. The reaction mixture wasthen thermally cycled at 68° C. for 6 minutes, 95° C. for 50 seconds andthen 55° C. for 50 seconds for 22 times. After cycling, the reactionmixture was held at 68° C. for 20 minutes and then cooled to 4° C. AfterDpnI digestion, an aliquot of the reaction mixture was used to transformXL10Gold cells, colonies picked, plasmids isolated (QIAprep® SpinMiniprep Kit, Qiagen) and the variants detected by restriction digests.Positives were checked for the correct sequence by DNA sequencing.

Oligonucleotide Primer Sequences:

Trypsin Loop 1A6 peptide 1A6FGF5-F: TCAATGCGCATGTGAAGAGATCTGGACTATGCTTTG(SEQ ID NO:68) CCGGTGTTCCGATATGCGTC 1A6FGF5-R:CGGAACACCGGCAAAGCATAGTCCAGATCTCTTCAC (SEQ ID NO:69)ATGCGCATTGATCGCAACAGG 1C2 peptide 1C2FGF5-F:TCAATGCGCATGTTGGCCCTTACTGTCAAAACATGC (SEQ ID NO:70) CGGTGTTCCGATATGCGTC1C2FGF5-F: CGGAACACCGGCATGTTTTGACAGTAAGGGCCCAAC (SEQ ID NO:71)ATGCGCATTGATCGCAACAGG 2E2 peptide 2E2FGF5-F:TCAATGCGCATGTCTTACAGTACTGTGGACTACATG (SEQ ID NO:72) CCGGTGTTCCGATATGCGTC2E2FGF5-R: CGGAACACCGGCATGTAGTCCACAGTACTGTAAGAC (SEQ ID NO:73)ATGCGCATGATCGCAACAGG 2E5 peptide 2E5FGF5-F:TCAATGCGCATGTACTCTTTGGAACAGATCTCCTTG (SEQ ID NO:74) CCGGTGTTCCGATATGCGTC2E5FGF5-R: CGGAACACCGGCAAGGAGATCTGTTCCAAAGAGTAC (SEQ ID NO:75)ATGCGCATTGATCGCAACAGG

Chymotrypsin Loop 1A6 peptide 1A6FGF5-2F:CAAAAGCTGTGCTTGTGAAGAGATCTGGACTATGCT (SEQ ID NO:76)TTGCTTTTGCGTCGACATCACGG 1A6FGF5-2R ACGCAAAAGCAAAGCATAGTCCAGATCTCTTCACAA(SEQ ID NO:77) GCACAGCTTTTGCATGCACTATG 1C2 peptide 1C2FGF5-2F:CAAAAGCTGTGCTTGTTGGGCCCTTACTGTCAAAAC (SEQ ID NO:78)ATGCTTTTGCGTCGACATCACGG 1C2FGF5-2R: ACGCAAAAGCATGTTTTGACAGTAAGGGCCCAACAA(SEQ ID NO:79) GCACAGCTTTTGCATGCACTATG 2E2 peptide 2E2FGF5-2F:CGGAACACCGGCATGTTTTGACAGTAAGGGCCCAAC (SEQ ID NO:80)ATGCGCATTGATCGCAACAGG 2E2FGF5-2R: ACGCAAAAGCATGTAGTCCACAGTACTGTAAGACAA(SEQ ID NO:81) GCACAGCTTTTGCATGCACTATG 2E5 peptide 2E5FGF5-2F:CAAAAGCTGTGCTTGTACTCTTTGGAATCGATCTCC (SEQ ID NO:82)TTGCTTTTGCGTCGACATCACGG 2E5FGF5-2R: ACGCAAAAGCAAGGAGATCGATTCCAAAGAGTACAA(SEQ ID NO:83) GCACAGCTTTTGCATGCACTATG

The sequences of the resulting constructs (SEQ ID NOS:84-101) areprovided in FIG. 9.

EXAMPLE 10 Cell-Based Assay Using NR6R-3T3 Cells

In this Example, experiments conducted to determine the biologicalactivity of the BLA-peptide fusion proteins, BBI/BCE-peptide fusionproteins, as well as BBI-peptide fusion proteins are described. Thebiological activity of the BLA-peptide fusion proteins was measured in aproliferation bioassay, using NR6R-3T3 cells. The NR6R-3T3 cell line isa cloned murine fibroblast cell line obtained from Dr. A. Rizzino. Thisassay system is well-defined and used by R & D Systems as a means tomeasure the activity of cytokines and growth factors (See, R&D Systems,Inc., protocols for more details). The cells are typically grown in DMEMcontaining 4.5 g/l glucose, supplemented with supplemented with 10% v/vFBS, 2 mM L-glutamine, 100 Units/ml penicillin, 100 μg/ml streptomycin.The Assay Medium used was DMEM, supplemented with 2% plasma-derivedbovine serum (PDS is dialyzed to remove growth factors), 2 mML-glutamine, 100 Units/ml penicillin, and 100 μg.ml streptomycin.

Cells were grown up and then incubated with the FGF-5-1 peptide fusionproteins (i.e., BLA-FGF-5, or BBI/BCE-FGF-5, with modifications to loop1 (“l1”) and/or loop 2 (“l2”), as described in Example 10. Cells wereharvested at confluency (approximately 3×10⁶ cells/flask). The standardssamples were diluted to working concentrations in PBS, 1.0 mg/ml BSA,and 0.1 μg/ml sodium heparin. Sodium heparin stock is in a solution ofDMEM+2% PDS. Prior to use, the microtiter plates were pre-blocked withPBS and 1% BSA for two hours at room temperature.

In order to measure the ability of the peptide fusions to “neutralize”or antagonize the bioactivity of the FGF-5 on the cells, constantconcentrations of FGF-5 were incubated with various concentrations ofthe peptide for either 30 minutes or 1 hour at room temperature or 37°C. in a 96-well microtiter plate. At 72 hours prior to setting up assay,cells were harvested at confluency (approximately 3×10⁶ cells/flask).Cells were plated in a 96-well flat bottom plate at 0.5×10⁴ cells in 100μl Growth Medium. The Growth Medium was removed after 48 hours andreplaced with 100 μl Assay Medium/well, so that cells would be in aresting phase and ready for assay, 24 hours later. The samples werediluted in DMEM-2% PDS. The standards and samples were then added to theplate. The assay mixture was incubated at 37° C. for approximately 16-18hours in a 5% CO₂ incubator with humidity. Then, 3H-thymidine (10μl/well of 25 μCi/ml ³H-thymidine working stock in Assay Medium) wasadded, for another 2 hours of incubation. The cells were subsequentlyharvested onto glass fiber filters. In order to ensure all attachedcells were removed, plate was rinsed with 200 μl 1× PBS without Ca or Mgand then treated with 100 μl 0.25% trypsin in EDTA. The amount of3H-thymidine incorporated into DNA was then determined. The doseresponse was then determined for the various concentrations of FGF-5.Some results obtained are provided in the Table of Peptides (See, above)and in FIGS. 7 and 12. As shown in FIG. 12, MM07-BBI and PS02-BBI bindand neutralize FGF-5 activity.

EXAMPLE 11 BioVeris Assay

In this Example, binding assays using BioVeris's protocols aredescribed. This assay system was optimized by testing several of thereagents at different concentrations to determine maximumsignal:background ratio. Various constructs produced as described abovewere tested in this assay system.

The assay was conducted as follows. First, the target (e.g., FGF-5) wasbiotinylated using EZ-Link Sulfo-NHS-Biotin (Pierce) and the protocolprovided by the manufacturer. The receptor (FGFrI(IIIc) was then taggedusing BV-tag ( BioVeris), using the BioVeris labeling protocol. Dilutedstock solutions of each reagent above (at optimized concentrations toprovide the best signal:background ratio) were prepared in PBS with0.05% TWEEN®-80 to the following concentrations: bFGF5: 500 ng/mL andBV-FGFrI(IIIc): 250 ng/mL.

Then, 50 μl of biotinylated target were added to each well of a 96 wellround bottom polypropylene plate (Costar 3365). Next, 50 μl of theconstruct to be tested (6 serial dilutions prepared (1000 nM→0) inPBS+0.05% TWEEN®-80, to provide a range of activity) were added to thewells. Then, 50 μl BV-FGFr(IIIc) diluted in PBS+0.05% TWEEN®-80 wasadded to the wells. The plate was incubated with gentle shaking (beingcareful to avoid well to well transfer) for 1 hour at room temperature.Following incubation, 50 μl streptavidin microbeads (Dynal) (0.2 mg/mLdiluted in PBS+0.05% TWEEN®-80) were added to the wells The plates werethen incubated for 30 minutes at room temperature, with gentle shaking.The total in the wells was then brought up to 250 μl/well with PBS+0.05%TWEEN®-80. The plate was then read on a BioVeris M Series 384 machine,using the default program. Some results obtained are provided in theTable of Peptides (See, above) and in FIG. 11.

EXAMPLE 12 In Vivo and Ex Vivo Assays

In this Example, in vivo assays to evaluate the constructs producedabove are described. In particular, in vivo assays are performed to testeach of the constructs for hair care, as known in the art (See e.g.,Tobin et al., J. Invest. Dermatol., 120:895 [2003]).

In additional embodiments, methods such as those that assess theinfluence of growth factors on hair growth find use in the presentinvention. Such methods are known in the art and include, but are notlimited to the Philpott assay system (See e.g., Philpott et al., J.Dermatol. Sci., 7 (Suppl.):S55-72 [1994]).

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It will be readily apparentto one skilled in the art that varying substitutions and modificationsmay be made to the invention disclosed herein without departing from thescope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the following claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

1. A composition comprising at least one peptide selected from the groupconsisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, and 14, wherein saidpeptide binds to a fibroblast growth factor.
 2. The composition of claim1, wherein said peptide is encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, and
 13. 3.The composition of claim 1, wherein said peptide is expressed in aprotease resistant scaffold.
 4. The composition of claim 3, wherein saidscaffold is a protease inhibitor.
 5. The composition of claim 4, whereinsaid protease inhibitor is selected from the group consisting ofBowman-Birk Inhibitor, soybean trypsin inhibitor, and Eglin chymotrypsininhibitor.
 6. The composition of claim 3, wherein said proteaseresistant scaffold and said peptide comprise a fusion protein.
 7. Acosmetic or pharmaceutical composition comprising said at least onepeptide of claim
 1. 8. The composition of claim 7, wherein saidcomposition is capable of modulating hair growth.
 9. The composition ofclaim 7, wherein said composition further comprises a scaffold.
 10. Amethod for modulating hair growth comprising: i) providing a compositioncomprising a peptide contained within a scaffold; ii) providing asubject to be treated; and iii) applying said composition to saidsubject in an area in which hair growth modulation is desired.
 11. Themethod of claim 10, wherein said peptide binds to a fibroblast growthfactor.
 12. The method of claim 11, wherein said fibroblast growthfactor (FGF) is FGF-5.
 13. The method of claim 10, wherein said scaffoldis selected from the group consisting of Bowman-Birk inhibitor, soybeantrypsin inhibitor, and Eglin chymotrypsin inhibitor.
 14. The method ofclaim 10, wherein said peptide is selected from the group consisting ofSEQ ID NOS: 2, 4, 6, 8, 10, 12, and
 14. 15. The method of claim 10,wherein said peptide is encoded by a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, and
 13. 16. Amethod for decreasing the activity of a fibroblast growth factorcomprising the steps of: i) providing a subject; and ii) administeringthe composition of claim 1 to said subject, under conditions such thatthe activity of said fibroblast growth factor is decreased.
 17. Themethod of claim 16, wherein said fibroblast growth factor is FGF-5. 18.A composition comprising an FGF-5 peptide sequence selected from thegroup of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 15-43, 45-65, 68-84, 86, 88,90, 92, 24, 26, 98, and 100.